Methods for amplifying nucleic acid using tag-mediated displacement

ABSTRACT

Disclosed are methods for amplifying a nucleic acid target region using an amplification oligomer comprising a target-binding segment and a heterologous displacer tag situated 5′ to the target-binding segment. Initiation of an amplification reaction from the tagged amplification oligomer produces an amplicon comprising the displacer tag, such that once the complement of the displacer tag has been incorporated into a second amplicon, a displacer oligonucleotide having a sequence substantially corresponding to the displacer tag sequence is used to participate in subsequent rounds of amplification for displacement of an extension product primed from a site within the second amplicon 5′ to the displacer priming site. Also disclosed are related kits and reaction mixtures comprising the displacer-tagged amplification oligomer and corresponding displacer oligonucleotide.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.16/195,702 filed Nov. 19, 2018, which is a continuation of U.S.application Ser. No. 14/928,796 filed Oct. 30, 2015 now U.S. Pat. No.10,214,760, which is a continuation of U.S. application Ser. No.14/344,372 filed Mar. 12, 2014 now U.S. Pat. No. 9,175,337, which is aNational Stage Entry of International Application No. PCT/US2012/056666filed Sep. 21, 2012, which claims the benefit under 35 USC 119(e) ofU.S. Provisional Application No. 61/537,452 filed Sep. 21, 2011, thecontents of each of which are incorporated herein by reference in theirentirety for all purposes.

REFERENCE TO SEQUENCE LISTING

This application includes an electronic sequence listing in a file named“693925SEQLST.TXT”, created Dec. 28, 2020 and containing 4,368 bytes,which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention resides in the technical fields of molecularbiology and diagnostics and relates in particular to amplification ofnucleic acids.

Background Art

The detection and/or quantitation of specific nucleic acid sequences isan important technique for identifying and classifying microorganisms,diagnosing infectious diseases, measuring response to various types oftreatment, and the like. Such procedures are also useful in detectingand quantitating microorganisms in foodstuffs, water, beverages,industrial and environmental samples, seed stocks, and other types ofmaterial where the presence of specific microorganisms may need to bemonitored.

Numerous amplification-based methods for the detection and quantitationof target nucleic acids are well-known and established in the art. Thepolymerase chain reaction, commonly referred to as PCR, uses multiplecycles of denaturation, annealing of primer pairs to opposite strands,and primer extension to exponentially increase copy numbers of thetarget sequence. (See, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, and4,800,159 to Mullis et al.; U.S. Pat. No. 5,804,375 to Gelfand et al.;Mullis et al., Meth. Enzymol. 155:335-350, 1987; and Murakawa et al.,DNA 7:287-295, 1988).

In a variation called RT-PCR, reverse transcriptase (RT) is used to makea complementary DNA (cDNA) from RNA, and the cDNA is then amplified byPCR to produce multiple copies of DNA. (See, e.g., U.S. Pat. Nos.5,322,770 and 5,310,652 to Gelfand et al.)

Another well-known amplification method is strand displacementamplification, commonly referred to as SDA, which uses cycles ofannealing pairs of primer sequences to opposite strands of a targetsequence, primer extension in the presence of a dNTP to produce a duplexhemiphosphorothioated primer extension product, endonuclease-mediatednicking of a hemimodified restriction endonuclease recognition site, andpolymerase-mediated primer extension from the 3′-end of the nick todisplace an existing strand and produce a strand for the next round ofprimer annealing, nicking and strand displacement, resulting ingeometric amplification of product. (See, e.g., Walker et al., Proc.Natl. Acad. Sci. USA 89:392-396, 1992; U.S. Pat. Nos. 5,270,184 and5,455,166 to Walker et al.; Walker et al., Nucleic Acids Research 20,1691-1696, 1992). Thermophilic SDA (tSDA) uses thermophilicendonucleases and polymerases at higher temperatures in essentially thesame method. (See, e.g., European Pat. No. 0 684 315.)

Other amplification methods include rolling circle amplification (RCA)(see, e.g., U.S. Pat. No. 5,854,033 to Lizardi); helicase dependentamplification (HDA) (see, e.g., Kong et al., U.S. Pat. Appln. Pub. No.US 2004-0058378 A1); and loop-mediated isothermal amplification (LAMP)(see, e.g., U.S. Pat. No. 6,410,278 to Notomi et al.).

Transcription-based amplification methods commonly used in the artinclude nucleic acid sequence based amplification, also referred to asNASBA (see, e.g., U.S. Pat. No. 5,130,238 to Malek et al.); methodswhich rely on the use of an RNA replicase to amplify the probe moleculeitself, commonly referred to as Qβ replicase (see, e.g., Lizardi et al.,BioTechnol. 6:1197-1202, 1988); transcription-based amplificationmethods (see, e.g., Kwoh et al., Proc. Natl. Acad. Sci. USA86:1173-1177, 1989) and self-sustained sequence replication (see, e.g.,Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-1878, 1990;Landgren, Trends in Genetics 9:199-202, 1993; and HELEN H. LEE et al.,Nucleic Acid Amplification Technologies (1997)).

Another transcription-based amplification method istranscription-mediated amplification, commonly referred to as TMA, whichsynthesizes multiple copies of a target nucleic acid sequenceautocatalytically under conditions of substantially constanttemperature, ionic strength, and pH, in which multiple RNA copies of thetarget sequence autocatalytically generate additional copies (see, e.g.,U.S. Pat. Nos. 5,480,784 and 5,399,491 to Kacian et al.). TMA is arobust and highly sensitive amplification system with demonstratedefficacy, which overcomes many of the problems associated with PCR-basedamplification systems. In particular, temperature cycling is notrequired.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for amplifyingnucleic acid target region. The method generally includes usingamplification oligonucleotide equipped with a heterologous displacer tagsituated 5′ to a target-binding segment and having a sequence thatsubstantially corresponds to the priming sequence of a displaceroligonucleotide. Initiation of an amplification reaction from the taggedamplification oligomer produces an amplicon comprising the displacertag. Once the complement of the displacer tag has been incorporated intoa second amplicon, thereby providing a displacer priming site, thedisplacer oligonucleotide participates in a subsequent round ofamplification for displacement of an extension product primed from asite within the second amplicon 5′ to the displacer priming site.

In some embodiments, the method of amplifying a nucleic acid targetregion uses a first forward amplification oligomer comprising (a) atarget-binding priming segment (T1) substantially complementary to a3′-end of the target region, (b) a first heterologous displacer tag (D1)located 5′ to T1, and (c) optionally, an intervening spacer segment (S1)between T1 and D1 (also referred to herein as a “T1-D1 forwardamplification oligomer”). The method generally comprises contacting atarget nucleic acid comprising the target region with (1) the firstamplification oligomer, wherein the contacting includes conditionswhereby the target nucleic acid serves as a template for extension fromthe first amplification oligomer to produce a first amplificationproduct comprising T1 and D1; (2) a second amplification oligomercomprising a target-binding segment T2 substantially complementary to aregion of the first amplicon that is the complement of a 5′-end of thetarget region, wherein the contacting further includes conditionswhereby the first amplicon serves as a template to produce a secondamplicon comprising segments cT1 and cD1, complementary to T1 and D1,respectively; (3) a third amplification oligomer comprisingtarget-binding priming segment T1_(p) having a nucleotide sequencesubstantially corresponding to T1, or substantially corresponding to thecomplement of a second amplicon target sequence cT1′ near or overlappingwith cT1 and situated 5′ to cD1; and (4) a fourth amplification oligomercomprising a displacer priming segment D1_(p) having a nucleotidesequence substantially corresponding to D1; wherein the contactingfurther includes conditions whereby the second amplicon serves as atemplate for extension from both the third and fourth amplificationoligomers, wherein extension of T1_(p) from a T1_(p):cT1 or T1_(p):cT1′hybrid produces a third amplicon, and wherein extension of D1_(p) from aD1_(p):cD1 hybrid produces a fourth amplicon while displacing the thirdamplicon.

In certain embodiments of the above method, the first amplificationoligomer further comprises a second heterologous displacer tag (D2)located 5′ to D1 and, optionally, a second intervening spacer segment(S2) between D1 and D2, such that the first amplicon further comprisesD2 and the second amplicon further comprises segment cD2, complementaryto D2. Typically, in such embodiments, the target nucleic acid isfurther contacted with (5) a fifth amplification oligomer comprising asecond displacer priming segment D2_(p) having a nucleotide sequencesubstantially corresponding to D2, under conditions whereby (i) thesecond amplicon serves as a template for extension from the fifthamplification oligomer, wherein extension of D2_(p) from a D2_(p):cD2hybrid produces a fifth amplicon comprising T1, D1, and D2_(p), and (ii)the fifth amplicon serves as a template for amplification from thesecond amplification oligomer to produce a sixth amplicon comprisingcT1, cD1, and cD2_(p). In a further variation, the target nucleic acidis also contacted with (6) a sixth amplification oligomer comprising (a)priming segment D1_(p) and (b) displacer tag D2 located 5′ to D1_(p),under conditions whereby (i) the fourth amplicon serves as a templatefor amplification from the second amplification oligomer to produce aseventh amplicon comprising segments cT1 and cD1_(p), (ii) at least oneof the second, sixth, and seventh amplicons serves as a template forextension from the sixth amplification oligomer, wherein extension ofD1_(p) from a D1_(p):cD1 or D1_(p):cD1_(p) hybrid produces an eighthamplicon comprising T1, D1_(p), and D2, and (iii) the eighth ampliconserves as a template for amplification from the second amplificationoligomer to produce a ninth amplicon comprising cT1, cD1_(p), and cD2.In some embodiments, the first amplification oligomer further comprisesa third heterologous displacer tag (D3) located 5′ to D2 and,optionally, a third intervening spacer segment (S3) between D2 and D3,whereby the first amplicon further comprises D3 and the second ampliconfurther comprises a segment cD3, complementary to D3; typically, inthese variations, the target nucleic acid is further contacted with (7)a seventh oligonucleotide amplification oligomer comprising a thirddisplacer priming segment D3_(p) having a nucleotide sequencesubstantially corresponding to D3, under conditions whereby (i) thesecond amplicon serves as a template for extension from the seventhamplification oligomer, wherein extension of D3_(p) from a D3_(p):cD3hybrid produces a tenth amplicon comprising T1, D1, D2, and D3_(p), and(ii) the tenth amplicon serves as a template for amplification from thesecond amplification oligomer to produce an eleventh amplicon comprisingcT1, cD1, cD2, and cD3_(p). In yet another variation, the target nucleicacid is further contacted with (8) an eighth amplification oligomercomprising (a) priming segment D2_(p) and (b) displacer tag D3 located5′ to D2_(p), under conditions whereby (i) at least one of the second,sixth, ninth, and eleventh amplicons serves as a template for extensionfrom the eighth amplification oligomer, wherein extension of D2_(p) froma D2_(p):cD2 or D2_(p):cD2_(p) hybrid produces a twelfth ampliconcomprising T1, D1 (or D1_(p)), D2_(p), and D3; and (ii) the twelfthamplicon serves as a template for amplification from the secondamplification oligomer to produce a thirteenth amplicon comprising cT1,cD1 (or cD1_(p)), cD2_(p), and cD3.

In other embodiments of the method using a T1-D1 forward amplificationoligomer as set forth above, the target nucleic acid is furthercontacted with (5) a fifth amplification oligomer comprising (a) primingsegment D1_(p) and (b) displacer tag D2 located 5′ to D1_(p); and (6) asixth amplification oligomer comprising a second displacer primingsegment D2_(p) having a nucleotide sequence substantially correspondingto D2. In such variations, the contacting typically includes conditionswhereby (i) the fourth amplicon serves as a template for amplificationfrom the second amplification oligomer to produce a fifth ampliconcomprising segments cT1 and cD1_(p), (ii) at least one of the second andfifth amplicons serves as a template for extension from the fifthamplification oligomer, wherein extension of D1_(p) from a D1_(p):cD1 orD1_(p):cD1_(p) hybrid produces a sixth amplicon comprising T1, D1_(p),and D2, (iii) the sixth amplicon serves as a template for amplificationfrom the second amplification oligomer to produce a seventh ampliconcomprising cT1, cD1_(p), and cD2, (iv) the seventh amplicon serves as atemplate for extension from the sixth amplification oligomer, whereinextension of D2_(p) from a D2_(p):cD2 hybrid produces an eighth ampliconcomprising T1, D1_(p), and D2_(p), and (v) the eighth amplicon serves asa template for amplification from the second amplification oligomer toproduce a ninth amplicon comprising cT1, cD1_(p), and cD2_(p). In afurther variation, the target nucleic acid is further contacted with (7)a seventh amplification oligomer comprising (a) priming segment D2_(p)and (b) displacer tag D3 located 5′ to D2_(p), and (8) an eightholigonucleotide amplification oligomer comprising a third displacerpriming segment D3_(p) having a nucleotide sequence substantiallycorresponding to D3. In such embodiments, the contacting typicallyincludes conditions whereby (i) at least one of the seventh and ninthamplicons serves as a template for extension from the seventhamplification oligomer, wherein extension of D2_(p) from a D2_(p):cD2 orD2_(p):cD2_(p) hybrid produces a tenth amplicon comprising T1, D1 (orD1_(p)), D2_(p), and D3, (ii) the tenth amplicon serves as a templatefor amplification from the second amplification oligomer to produce aneleventh amplicon comprising cT1, cD1 (or cD1_(p)), cD2_(p), and cD3,(iii) the eleventh amplicon serves as a template for extension from theeighth amplification oligomer, wherein extension of D3_(p) from aD3_(p):cD3 hybrid produces a twelfth amplicon comprising T1, D1, D2, andD3_(p), and (iv) the twelfth amplicon serves as a template foramplification from the second amplification oligomer to produce athirteenth amplicon comprising cT1, cD1, cD2, and cD3_(p).

In any of the above embodiments, the second amplification oligomer mayfurther comprise a fourth heterologous displacer tag (D4) located 5′ toT2 and, optionally, a fourth intervening spacer segment (S4) between T2and D4, such that the second amplicon comprises T2 and D4, and each ofthe third and fourth amplicons comprises segments cT2 and cD4,complementary to T2 and D4, respectively. In such variations, the targetnucleic acid is further contacted with (9) a ninth amplificationoligomer comprising a priming segment T2_(p) having a nucleotidesequence substantially corresponding to T2, or substantiallycorresponding to the complement of a third or fourth amplicon targetsequence cT2′ near or overlapping with cT2 and situated 5′ to cD4; and(10) a tenth amplification oligomer comprising a fourth displacerpriming segment D4_(p) having a nucleotide sequence substantiallycorresponding to D4, under conditions whereby at least one of the thirdand fourth amplicons serves as a template for extension from both theninth and tenth amplification oligomers, wherein extension of T2_(p)from a T2_(p):cT2 or T2_(p):cT2′ hybrid produces a fourteenth amplicon,and wherein extension of D4_(p) from a D4_(p):cD4 hybrid produces afifteenth amplicon while displacing the fourteenth amplicon.

In certain embodiments of the method as above, the affinity of D1_(p)for its complement is lower than that of T1. In other embodimentscomprising the use of a D2_(p) displacer oligomer, (a) the affinity ofD1_(p) for its complement is lower than that of T1 and/or (b) theaffinity of D2_(p) for its complement is lower than that of D1_(p). Inyet other embodiments comprising the use of a D3_(p) displacer oligomer,(a) the affinity of D1_(p) for its complement is lower than that of T1and/or (b) the affinity of D2_(p) for its complement is lower than thatof D1_(p) and/or (c) the affinity of D3_(p) for its complement is lowerthan that of D2_(p).

In some embodiments of the method as above, the target nucleic acid isRNA. In some such embodiments, extension from the first amplificationoligomer comprises contacting the target nucleic acid with a reversetranscriptase (RT). In more particular variations, the secondamplification oligomer further comprises an RNA polymerase promotersequence (P) (e.g., a T7 promoter sequence) located 5′ to T2, such thateach of the third and fourth amplicons comprises a segment cP,complementary to the promoter sequence; and contacting the targetnucleic acid further includes conditions whereby an RNA polymeraseinitiates transcription upon recognizing a double stranded promotersequence (P:cP) formed by extension of either the third or fourthamplification oligomer on the second amplicon, thereby producing an RNAamplicon. In a related variation, the second amplification oligomerfurther comprises an RNA polymerase promoter sequence (P) (e.g., a T7promoter sequence) located 5′ to T2 and is modified to prevent theinitiation of DNA synthesis from its 3′-end, and the target nucleic acidis further contacted with a terminating oligonucleotide comprising atarget-binding sequence substantially complementary to a target sequencethat is adjacent to the 5′-end of the target region. In theseembodiments, contacting the target nucleic acid typically includesconditions whereby extension of the first amplification oligomer isterminated at the 3′-end of the terminating oligonucleotide, therebyproviding a 3′-end for the first amplicon that corresponds to the 5′-endof the target region; the promoter sequence P of the secondamplification oligonucleotide serves as a template for extension fromthe 3′-end of the first amplicon, whereby the first amplicon comprises asegment cP, complementary to the promoter sequence, thereby forming adouble stranded promoter sequence (P:cP); and an RNA polymeraseinitiates transcription upon recognizing the double stranded promotersequence, thereby producing an RNA amplicon as the second amplicon.

In some embodiments, the method of amplifying a nucleic acid targetregion uses a second reverse amplification oligomer comprising (a) atarget-binding segment T2 substantially complementary to a region of afirst amplicon that is the complement of a 5′-end of the target region,(b) a first heterologous displacer tag (D1) located 5′ to T2, and (c)optionally, an intervening spacer segment (S1) between T2 and D1 (alsoreferred to herein as a “T2-D1 reverse amplification oligomer”). Themethod generally comprises contacting a target nucleic acid comprisingthe target region with (1) a first amplification oligomer comprising atarget-binding priming segment (T1) substantially complementary to a3′-end of the target region; wherein the contacting includes conditionswhereby the target nucleic acid serves as a template for extension fromthe first amplification oligomer to produce a first amplicon; (2) thesecond amplification oligomer, wherein the contacting further includesconditions whereby the first amplicon serves as a template foramplification from the second amplification oligomer to produce a secondamplicon comprising T2 and D1, and whereby the second amplicon serves asa template for extension from the first amplification oligomer toproduce a third amplicon comprising segments cT2 and cD1, complementaryto T2 and D1, respectively; (3) a third amplification oligomercomprising target-binding priming segment T2_(p) having a nucleotidesequence substantially corresponding to T2, or substantiallycorresponding to the complement of a third amplicon target sequence cT2′near or overlapping with cT2 and situated 5′ to cD1; and (4) a fourthamplification oligomer comprising a displacer priming segment D1_(p)having a nucleotide sequence substantially corresponding to D1; whereinthe contacting further includes conditions whereby the third ampliconserves as a template for extension from both the third and fourthamplification oligomers, wherein extension of T2_(p) from a T2_(p):cT2or T2_(p):cT2′ hybrid produces a fourth amplicon, and wherein extensionof D1_(p) from a D1_(p):cD1 hybrid produces a fifth amplicon whiledisplacing the fourth amplicon.

In certain embodiments of the above method using a T2-D1 reverseamplification oligomer, the second (T2-D1 reverse) amplificationoligomer further comprises a second heterologous displacer tag (D2)located 5′ to D1 and, optionally, a second intervening spacer segment(S2) between D1 and D2, such that the second amplicon further comprisesD2 and the third amplicon further comprises segment cD2, complementaryto D2. Typically, in such embodiments, the target nucleic acid isfurther contacted with (5) a fifth amplification oligomer comprising asecond displacer priming segment D2_(p) having a nucleotide sequencesubstantially corresponding to D2, under conditions whereby (i) thethird amplicon serves as a template for extension from the fifthamplification oligomer, wherein extension of D2_(p) from a D2_(p):cD2hybrid produces a sixth amplicon comprising T2, D1, and D2_(p), and (ii)the sixth amplicon serves as a template for extension from the firstamplification oligomer to produce a seventh amplicon comprising cT2,cD1, and cD2_(p). In a further variation, the target nucleic acid isalso contacted with (6) a sixth amplification oligomer comprising (a)priming segment D1_(p) and (b) displacer tag D2 located 5′ to D1_(p),under conditions whereby (i) the fifth amplicon serves as a template forextension from the first amplification oligomer to produce an eighthamplicon comprising segments cT2 and cD1_(p), (ii) at least one of thethird, seventh, and eighth amplicons serves as a template for extensionfrom the sixth amplification oligomer, wherein extension of D1_(p) froma D1_(p):cD1 or D1_(p):cD1_(p) hybrid produces a ninth ampliconcomprising T2, D1_(p), and D2, and (iii) the ninth amplicon serves as atemplate for extension from the first amplification oligomer to producea tenth amplicon comprising cT2, cD1_(p), and cD2. In some embodiments,the second amplification oligomer further comprises a third heterologousdisplacer tag (D3) located 5′ to D2 and, optionally, a third interveningspacer segment (S3) between D2 and D3, whereby the second ampliconfurther comprises D3 and the third amplicon further comprises a segmentcD3, complementary to D3; in these variations, the target nucleic acidis further contacted with (7) a seventh amplification oligomercomprising a third displacer priming segment D3_(p) having a nucleotidesequence substantially corresponding to D3, under conditions whereby (i)the third amplicon serves as a template for extension from the seventhamplification oligomer, wherein extension of D3_(p) from a D3_(p):cD3hybrid produces an eleventh amplicon comprising T2, D1, D2, and D3_(p),and (ii) the eleventh amplicon serves as a template for extension fromthe first amplification oligomer to produce a twelfth ampliconcomprising cT2, cD1, cD2, and cD3_(p). In yet a another variation, thetarget nucleic acid is further contacted with (8) an eighthamplification oligomer comprising (a) priming segment D2_(p) and (b)displacer tag D3 located 5′ to D2_(p), under conditions whereby (i) atleast one of the third, seventh, tenth, and twelfth amplicons serves asa template for extension from the eighth amplification oligomer, whereinextension of D2_(p) from a D2_(p):cD2 or D2_(p):cD2_(p) hybrid producesa thirteenth amplicon comprising T2, D1 (or D1_(p)), D2_(p), and D3, and(ii) the thirteenth amplicon serves as a template for extension from thefirst amplification oligomer to produce a fourteenth amplicon comprisingcT2, cD1 (or cD1_(p)), cD2_(p), and cD3.

In other embodiments of the method using a T2-D1 reverse amplificationoligomer as set forth above, the target nucleic acid is furthercontacted with (5) a fifth amplification oligomer comprising (a) primingsegment D1_(p) and (b) displacer tag D2 located 5′ to D1_(p); and (6) asixth amplification oligomer comprising a second displacer primingsegment D2_(p) having a nucleotide sequence substantially correspondingto D2. In such variations, the contacting typically includes conditionswhereby (i) the fifth amplicon serves as a template for extension fromthe first amplification oligomer to produce a sixth amplicon comprisingsegments cT2 and cD1_(p), (ii) at least one of the third and sixthamplicons serves as a template for extension from the fifthamplification oligomer, wherein extension of D1_(p) from a D1_(p):cD1 orD1_(p):cD1_(p) hybrid produces a seventh amplicon comprising T2, D1_(p),and D2, (iii) the seventh amplicon serves as a template for extensionfrom the first amplification oligomer to produce an eighth ampliconcomprising cT2, cD1_(p), and cD2, (iv) the eighth amplicon serves as atemplate for extension from the sixth amplification oligomer, whereinextension of D2_(p) from a D2_(p):cD2 hybrid produces a ninth ampliconcomprising T2, D1, and D2_(p), and (v) the ninth amplicon serves as atemplate for extension from the first amplification oligomer to producea tenth amplicon comprising cT2, cD1, and cD2_(p). In a furthervariation, the target nucleic acid is further contacted with (7) aseventh amplification oligomer comprising (a) priming segment D2_(p) and(b) displacer tag D3 located 5′ to D2_(p), and (8) an eighthamplification oligomer comprising a third displacer priming segmentD3_(p) having a nucleotide sequence substantially corresponding to D3.In such embodiments, the contacting typically includes conditionswhereby (i) at least one of the eighth and tenth amplicons serves as atemplate for extension from the seventh amplification oligomer, whereinextension of D2_(p) from a D2_(p):cD2 or D2_(p):cD2_(p) hybrid producesan eleventh amplicon comprising T2, D1 (or D1_(p)), D2_(p), and D3, (ii)the eleventh amplicon serves as a template for extension from the firstamplification oligomer to produce a twelfth amplicon comprising cT2, cD1(or cD1_(p)), cD2_(p), and cD3, (iii) the twelfth amplicon serves as atemplate for extension from the eighth amplification oligomer, whereinextension of D3_(p) from a D3_(p):cD3 hybrid produces an thirteenthamplicon comprising T2, D1, D2, and D3_(p), and (iv) the thirteenthamplicon serves as a template for extension from the first amplificationoligomer to produce a fourteenth amplicon comprising cT2, cD1, cD2, andcD3_(p).

In any of the above embodiments, the first amplification oligomer mayfurther comprise a fourth heterologous displacer tag (D4) located 5′ toT1 and, optionally, a fourth intervening spacer segment (S4) between T1and D4, such that the first amplicon comprises T1 and D4, and the secondamplicon comprises segments cT1 and cD4, complementary to T1 and D4,respectively. In such variations, the target nucleic acid is furthercontacted with a (9) a ninth amplification oligomer comprising a primingsegment T1_(p) having a nucleotide sequence substantially correspondingto T1, or substantially corresponding to the complement of a secondamplicon target sequence cT1′ near or overlapping with cT1 and situated5′ to cD4; and (10) a tenth amplification oligomer comprising a fourthdisplacer priming segment D4_(p) having a nucleotide sequencesubstantially corresponding to D4, under conditions whereby the secondamplicon serves as a template for extension from both the ninth andtenth amplification oligomers, wherein extension of T1_(p) from aT1_(p):cT1 or T1_(p):cT1′ hybrid produces a fifteenth amplicon, andwherein extension of D4_(p) from a D4_(p):cD4 hybrid produces asixteenth amplicon while displacing the fifteenth amplicon.

In certain embodiments of the method using a T2-D1 reverse amplificationoligomer as set forth above, the affinity of D1_(p) for its complementis lower than that of T1. In other embodiments comprising the use of aD2p displacer oligomer, (a) the affinity of D1_(p) for its complement islower than that of T2 and/or (b) the affinity of D2_(p) for itscomplement is lower than that of D1_(p). In yet other embodimentscomprising the use of a D3p displacer oligomer, (a) the affinity ofD1_(p) for its complement is lower than that of T2 and/or (b) theaffinity of D2_(p) for its complement is lower than that of D1_(p)and/or (c) the affinity of D3_(p) for its complement is lower than thatof D2_(p).

In some embodiments of the method using a T2-D1 reverse amplificationoligomer as set forth above, the target nucleic acid is RNA. In somesuch embodiments, extension from the first amplification oligomercomprises contacting the target nucleic acid with a reversetranscriptase (RT). In more particular variations, the firstamplification oligomer further comprises an RNA polymerase promotersequence (P) (e.g., at T7 promoter sequence) located 5′ to T1, such thatthe second amplicon comprises a segment cP, complementary to thepromoter sequence; and contacting the target nucleic acid furtherincludes conditions whereby an RNA polymerase initiates transcriptionupon recognizing a double-stranded promoter sequence (P:cP) formed byextension of the second amplification oligomer on the first amplicon,thereby producing an RNA amplicon.

In more particular embodiments of the above method utilizing either aT1-D1 forward amplification oligomer or a T2-D1 reverse amplificationoligomer and further comprising a D2 displacer tag, D1 and D2 aredifferent (i.e., have different nucleotide sequences); in alternativeembodiments, D1 and D2 are the same. In more particular embodiments ofthe above method further comprising a D3 displacer tag, at least two(e.g., all three) of D1, D2, and D3 are different; in alternativeembodiments, at least two (e.g., all three) of D1, D2, and D3 are thesame.

In other embodiments, the method of amplifying a nucleic acid targetregion uses a first forward amplification oligomer comprising (a) atarget-binding priming segment (T1) substantially complementary to a3′-end of the target region, (b) a heterologous universal tag (U1)located 5′ to T1, (c) a first heterologous displacer tag (D1) located 5′to U1, and (d) optionally, an intervening spacer segment (S1) between U1and D1 (also referred to herein as a “T1-U1-D1 forward amplificationoligomer”). The method generally comprises contacting a target nucleicacid comprising the target region with (1) the first amplificationoligomer, wherein the contacting includes conditions whereby the targetnucleic acid serves as a template for extension from the firstamplification oligomer to produce a first amplicon comprising U1 and D1;(2) a second amplification oligomer comprising (a) a target-bindingsegment T2 substantially complementary to a region of the first ampliconthat is the complement of a 5′-end of the target region and (b)optionally, a heterologous universal tag (U2) located 5′ to T2, whereinthe contacting further includes conditions whereby the first ampliconserves as a template for amplification from the second amplificationoligomer to produce a second amplicon comprising segments cU1 and cD1,complementary to U1 and D1, respectively, and optionally comprising U2;(3) a third amplification oligomer comprising a universal primingsegment U1_(p) having a nucleotide sequence substantially correspondingto U1; (4) a fourth amplification oligomer comprising a displacerpriming segment D1_(p) having a nucleotide sequence substantiallycorresponding to D1; and (5) if the second amplification oligomercomprises U2, a fifth amplification oligomer comprising a seconduniversal priming segment U2_(p) having a nucleotide sequencesubstantially corresponding to U2; wherein the contacting furtherincludes conditions whereby the second amplicon serves as a template forextension from both the third and fourth amplification oligomers,wherein extension of U1_(p) from a U1_(p):cU1 hybrid produces a thirdamplicon, and wherein extension of D1_(p) from a D1_(p):cD1 hybridproduces a fourth amplicon while displacing the third amplicon.

In certain embodiments of the above method utilizing a T1-U1-D1 forwardamplification oligomer, the first amplification oligomer furthercomprises a second heterologous displacer tag (D2) located 5′ to D1 and,optionally, a second intervening spacer segment (S2) between D1 and D2,such that the first amplicon further comprises D2 and the secondamplicon further comprises segment cD2, complementary to D2. Typically,in such embodiments, the target nucleic acid is further contacted with(6) a sixth amplification oligomer comprising a second displacer primingsegment D2_(p) having a nucleotide sequence substantially correspondingto D2, wherein the contacting includes conditions whereby (i) the secondamplicon serves as a template for extension from the sixth amplificationoligomer, wherein extension of D2_(p) from a D2_(p):cD2 hybrid producesa fifth amplicon comprising U1, D1, and D2_(p), and (ii) the fifthamplicon serves as a template for amplification from the second or fifthamplification oligomer to produce a sixth amplicon comprising cU1, cD1,and cD2_(p). In a further variation, the target nucleic acid is alsocontacted with (7) a seventh amplification oligomer comprising (a)priming segment D1_(p) and (b) displacer tag D2 located 5′ toD1_(p),under conditions whereby (i) the fourth amplicon serves as atemplate for amplification from the second or fifth amplificationoligomer to produce a seventh amplicon comprising segments cT1 andcD1_(p), (ii) at least one of the second, sixth, and seventh ampliconsserves as a template for extension from the seventh amplificationoligomer, wherein extension of D1_(p) from a D1_(p):cD1 orD1_(p):cD1_(p) hybrid produces an eighth amplicon comprising U1, D1_(p),and D2, and (iii) the eighth amplicon serves as a template foramplification from the second or fifth amplification oligomer to producea ninth amplicon comprising cU1, cD1_(p), and cD2. In some embodiments,the first amplification oligomer further comprises a third heterologousdisplacer tag (D3) located 5′ to D2 and, optionally, a third interveningspacer segment (S3) between D2 and D3, whereby the first ampliconfurther comprises D3 and the second amplicon further comprises a segmentcD3, complementary to D3; typically, in these variations, the targetnucleic acid is further contacted with (8) an eighth amplificationoligomer comprising a third displacer priming segment D3_(p) having anucleotide sequence substantially corresponding to D3, under conditionswhereby (i) the second amplicon serves as a template for extension fromthe eighth amplification oligomer, wherein extension of D3_(p) from aD3_(p):cD3 hybrid produces a tenth amplicon comprising U1, D1, D2, andD3_(p); and (ii) the tenth amplicon serves as a template foramplification from the second or fifth amplification oligomer to producean eleventh amplicon comprising cU1, cD1, cD2, and cD3_(p). In yetanother variation, the target nucleic acid is further contacted with (9)a ninth amplification oligomer comprising (a) priming segment D2_(p) and(b) displacer tag D3 located 5′ to D2_(p), under conditions whereby (i)at least one of the second, sixth, ninth, and eleventh amplicons servesas a template for extension from the seventh amplification oligomer,wherein extension of D2_(p) from a D2_(p):cD2 or D2_(p):cD2_(p) hybridproduces a twelfth amplicon comprising U1, D1 (or D1_(p)), D2p, and D3,and (ii) the twelfth amplicon serves as a template for amplificationfrom the second or fifth amplification oligomer to produce a thirteenthamplicon comprising cU1, cD1 (or cD1_(p)), cD2_(p), and cD3.

In other embodiments of the method using a T1-U1-D1 forwardamplification oligomer as set forth above, the target nucleic acid isfurther contacted with (6) a sixth amplification oligomer comprising (a)priming segment D1_(p) and (b) displacer tag D2 located 5′ to D1_(p),and (7) a seventh amplification oligomer comprising a second displacerpriming segment D2_(p) having a nucleotide sequence substantiallycorresponding to D2. In such variations, the contacting typicallyincludes conditions whereby (i) the fourth amplicon serves as a templatefor amplification from the second or fifth amplification oligomer toproduce a fifth amplicon comprising segments cU1 and cD1_(p), (ii) atleast one of the second and fifth amplicons serves as a template forextension from the sixth amplification oligomer, wherein extension ofD1_(p) from a D1_(p):cD1 or D1_(p):cD1_(p) hybrid produces a sixthamplicon comprising U1, D1_(p), and D2, (iii) the sixth amplicon servesas a template for amplification from the second amplification oligomerto produce a seventh amplicon comprising cU1, cD1_(p), and cD2; (iv) theseventh amplicon serves as a template for extension from the seventhamplification oligomer, wherein extension of D2_(p) from a D2_(p):cD2hybrid produces an eighth amplicon comprising U1, D1_(p), and D2_(p),and (v) the eighth amplicon serves as a template for amplification fromthe second or fifth amplification oligomer to produce a ninth ampliconcomprising cU1, cD1_(p), and cD2_(p). In a further variation, the targetnucleic acid is further contacted with (8) an eighth amplificationoligomer comprising (a) priming segment D2_(p) and (b) displacer tag D3located 5′ to D2_(p), and (9) a ninth oligonucleotide amplificationoligomer comprising a third displacer priming segment D3_(p) having anucleotide sequence substantially corresponding to D3. In suchembodiments, the contacting typically includes conditions whereby (i) atleast one of the seventh and ninth amplicons serves as a template forextension from the eighth amplification oligomer, wherein extension ofD2_(p) from a D2_(p):cD2 or D2_(p):cD2_(p) hybrid produces a tenthamplicon comprising U1, D1/D1_(p), D2_(p), and D3, (ii) the tenthamplicon serves as a template for amplification from the second or fifthamplification oligomer to produce an eleventh amplicon comprising cU1,cD1 (or cD1_(p)), cD2_(p), and cD3, (iii) the eleventh amplicon servesas a template for extension from the ninth amplification oligomer,wherein extension of D3_(p) from a D3_(p):cD3 hybrid produces a twelfthamplicon comprising U1, D1, D2, and D3_(p), and (iv) the twelfthamplicon serves as a template for amplification from the second or fifthamplification oligomer to produce a thirteenth amplicon comprising cU1,cD1, cD2, and cD3_(p).

In any of the above embodiments using a T1-U1-D1 forward amplificationoligomer, the second amplification oligomer may further comprise afourth heterologous displacer tag (D4) located 5′ to T2 and, optionally,a fourth intervening spacer segment between T2 and D4, whereby thesecond amplicon comprises T2 and D4; and whereby each of the third andfourth amplicons comprises segments cT2 and cD4, complementary to T2 andD4, respectively. Typically, in such variations, the target nucleic acidis further contacted with (10) a tenth amplification oligomer comprisinga priming segment T2_(p) having a nucleotide sequence substantiallycorresponding to T2, or substantially corresponding to the complement ofa third or fourth amplicon target sequence cT2′ near or overlapping withcT2 and situated 5′ to cD4, and (11) an eleventh amplification oligomercomprising a fourth displacer priming segment D4_(p) having a nucleotidesequence substantially corresponding to D4, under conditions whereby atleast one of the third and fourth amplicons serves as a template forextension from both the tenth and eleventh amplification oligomers,wherein extension of T2_(p) from a T2_(p):cT2 or T2_(p):cT2′ hybridproduces a fourteenth amplicon, and wherein extension of D4_(p) from aD4_(p):cD4 hybrid produces a fifteenth amplicon while displacing thefourteenth amplicon.

In other embodiments of the method using a T1-U1-D1 forwardamplification oligomer as set forth above, the second amplificationoligomer comprises U2 and further comprises a fourth heterologousdisplacer tag (D4) located 5′ to U2 and, optionally, a fourthintervening spacer segment (S4) between U2 and D4, whereby the secondamplicon comprises U2 and D4; and whereby each of the third and fourthamplicons comprises segments cU2 and cD4, complementary to U2 and D4,respectively. Typically, in such variations, the target nucleic acid isfurther contacted with (10) a tenth amplification oligomer comprising afourth displacer priming segment D4_(p) having a nucleotide sequencesubstantially corresponding to D4, under conditions whereby at least oneof the third and fourth amplicons serves as a template for extensionfrom both the fifth and tenth amplification oligomers, wherein extensionof U2_(p) from a U2_(p):cU2 hybrid produces a fourteenth amplicon, andwherein extension of D4_(p) from a D4_(p):cD4 hybrid produces afifteenth amplicon while displacing the fourteenth amplicon.

In certain embodiments of the method using a T1-U1-D1 forwardamplification oligomer as set forth above, the affinity of D1_(p) forits complement is lower than that of U1. In other embodiments comprisingthe use of a D2p displacer oligomer, (a) the affinity of D1_(p) for itscomplement is lower than that of U1 and/or (b) the affinity of D2_(p)for its complement is lower than that of D1_(p). In yet otherembodiments comprising the use of a D3_(p) displacer oligomer, (a) theaffinity of D1_(p) for its complement is lower than that of U1 and/or(b) the affinity of D2_(p) for its complement is lower than that ofD1_(p) and/or (c) the affinity of D3_(p) for its complement is lowerthan that of D2_(p).

In some embodiments of the method using a T1-U1-D1 forward amplificationoligomer as set forth above, the target nucleic acid is RNA. In somesuch embodiments, extension from the first amplification oligomercomprises contacting the target nucleic acid with a reversetranscriptase (RT). In more particular variations, the secondamplification oligomer further comprises an RNA polymerase promotersequence (P) (e.g., a T7 promoter sequence) located 5′ to T2, such thateach of the third and fourth amplicons comprises a segment cP,complementary to the promoter sequence; and contacting the nucleic acidfurther comprises conditions whereby an RNA polymerase initiatestranscription upon recognizing a double-stranded promoter sequence(P:cP) formed by extension of either the third or fourth amplificationoligomer on the second amplicon, thereby producing an RNA amplicon. In arelated variation, the second amplification oligomer further comprisesan RNA polymerase promoter sequence (P) (e.g., a T7 promoter sequence)located 5′ to T2 and is modified to prevent the initiation of DNAsynthesis from its 3′-end, and the target nucleic acid is furthercontacted with a terminating oligonucleotide comprising a target-bindingsequence substantially complementary to a target sequence that isadjacent to the 5′-end of the target region. In these embodiments,contacting the target nucleic acid typically includes conditions wherebyextension of the first amplification oligomer is terminated at the3′-end of the terminating oligonucleotide, thereby providing a 3′-endfor the first amplicon that corresponds to the 5′-end of the targetregion; the promoter sequence P of the second amplificationoligonucleotide serves as a template for extension from the 3′-end ofthe first amplicon, whereby the first amplicon comprises a segment cP,complementary to the promoter sequence, thereby forming a doublestranded promoter sequence (P:cP); and an RNA polymerase initiatestranscription upon recognizing the double stranded promoter sequence,thereby producing an RNA amplicon as the second amplicon.

In some embodiments, the method of amplifying a nucleic acid targetregion uses a second reverse amplification oligomer comprising (a) atarget-binding segment T2 substantially complementary to a region of thefirst amplicon that is the complement of a 5′-end of the target region,(b) a heterologous universal tag (U1) located 5′ to T2, (c) a firstheterologous displacer tag (D1) located 5′ to U1, and (d) optionally, anintervening spacer segment (S1) between U1 and D1 (also referred toherein as a “T2-U1-D1 reverse amplification oligomer”). The methodgenerally comprises contacting a target nucleic acid comprising thetarget region with (1) a first amplification oligomer comprising (a) atarget-binding priming segment (T1) substantially complementary to a3′-end of the target region, and (b) optionally, a heterologousuniversal tag (U2) located 5′ to T1; wherein the contacting includesconditions whereby the target nucleic acid serves as a template forextension from the first amplification oligomer to produce a firstamplicon; (2) the second amplification oligomer comprising, wherein thecontacting further includes conditions whereby the first amplicon servesas a template for amplification from the second amplification oligomerto produce a second amplicon comprising U1 and D1, and whereby thesecond amplicon serves as a template for extension from the firstamplification oligomer to produce a third amplicon comprising segmentscU1 and cD1, complementary to U1 and D1, respectively; (3) a thirdamplification oligomer comprising a universal priming segment U1_(p)having a nucleotide sequence substantially corresponding to U1; (4) afourth amplification oligomer comprising a displacer priming segmentD1_(p) having a nucleotide sequence substantially corresponding to D1;and (5) if the first amplification oligomer comprises U2, a fifthamplification oligomer comprising a second universal priming segmentU2_(p) having a nucleotide sequence substantially corresponding to U2;wherein the contacting further includes conditions whereby the thirdamplicon serves as a template for extension from both the third andfourth amplification oligomers, wherein extension of U1_(p) from aU1_(p):cU1 hybrid produces a fourth amplicon, and wherein extension ofD1_(p) from a D1_(p):cD1 hybrid produces a fifth amplicon whiledisplacing the fourth amplicon.

In certain embodiments of the above method using a T2-U1-D1 reverseamplification oligomer, the second (T2-U1-D1 reverse) amplificationoligomer further comprises a second heterologous displacer tag (D2)located 5′ to D1 and, optionally, a second intervening spacer segment(S2) between D1 and D2, whereby the second amplicon further comprises D2and the third amplicon further comprises segment cD2, complementary toD2. Typically, in such embodiments, the target nucleic acid is furthercontacted with (6) a sixth amplification oligomer comprising a seconddisplacer priming segment D2_(p) having a nucleotide sequencesubstantially corresponding to D2, under conditions whereby (i) thethird amplicon serves as a template for extension from the sixthamplification oligomer, wherein extension of D2_(p) from a D2_(p):cD2hybrid produces a sixth amplicon comprising U1, D1, and D2_(p), and (ii)the sixth amplicon serves as a template for extension from the first orfifth amplification oligomer to produce a seventh amplicon comprisingcU1, cD1, and cD2_(p). In a further variation, the target nucleic acidis also contacted with (7) a seventh amplification oligomer comprising(a) priming segment D1_(p) and (b) displacer tag D2 located 5′ toD1_(p), under conditions whereby (i) the fifth amplicon serves as atemplate for extension from the first or fifth amplification oligomer toproduce an eighth amplicon comprising segments cU1 and cD1_(p), (ii) atleast one of the third, seventh, and eighth amplicons serves as atemplate for extension from the seventh amplification oligomer, whereinextension of D1_(p) from a D1_(p):cD1 or D1_(p):cD1_(p) hybrid producesa ninth amplicon comprising U1, D1_(p), and D2, and (iii) the ninthamplicon serves as a template for extension from the first or fifthamplification oligomer to produce a tenth amplicon comprising cU1,cD1_(p), and cD2. In some embodiments, the second amplification oligomerfurther comprises a third heterologous displacer tag (D3) located 5′ toD2 and, optionally, a third intervening spacer segment (S3) between D2and D3, whereby the second amplicon further comprises D3 and the thirdamplicon further comprises a segment cD3, complementary to D3; andwherein the target nucleic acid is further contacted with (8) an eighthamplification oligomer comprising a third displacer priming segmentD3_(p) having a nucleotide sequence substantially corresponding to D3,under conditions whereby (i) the third amplicon serves as a template forextension from the eighth amplification oligomer, wherein extension ofD3_(p) from a D3_(p):cD3 hybrid produces an eleventh amplicon comprisingU1, D1, D2, and D3_(p), (ii) the eleventh amplicon serves as a templatefor extension from the first or fifth amplification oligomer to producean twelfth amplicon comprising cU1, cD1, cD2, and cD3_(p). In yetanother variation, the target nucleic acid is further contacted with (9)a ninth amplification oligomer comprising (a) priming segment D2_(p) and(b) displacer tag D3 located 5′ to D2_(p), under conditions whereby (i)at least one of the third, seventh, tenth, and twelfth amplicons servesas a template for extension from the eighth amplification oligomer,wherein extension of D2_(p) from a D2_(p):cD2 or D2_(p):cD2_(p) hybridproduces a thirteenth amplicon comprising U1, D1 (or D1_(p)), D2_(p),and D3, and (ii) the thirteenth amplicon serves as a template forextension from the first or fifth amplification oligomer to produce afourteenth amplicon comprising cU1, cD1 (or cD1_(p)), cD2_(p), and cD3.

In other embodiments of the method using a T2-U1-D1 reverseamplification oligomer as set forth above, the target nucleic acid isfurther contacted with (6) a sixth amplification oligomer comprising (a)priming segment D1_(p) and (b) displacer tag D2 located 5′ to D1_(p),and (7) a seventh amplification oligomer comprising a second displacerpriming segment D2_(p) having a nucleotide sequence substantiallycorresponding to D2. In such variations, the contacting includesconditions whereby (i) the fifth amplicon serves as a template forextension from the first or fifth amplification oligomer to produce asixth amplicon comprising segments cU2 and cD1_(p), (ii) at least one ofthe third and sixth amplicons serves as a template for extension fromthe sixth amplification oligomer, wherein extension of D1_(p) from aD1_(p):cD1 or D1_(p):cD1_(p) hybrid produces a seventh ampliconcomprising U2, D1_(p), and D2, (iii) the seventh amplicon serves as atemplate for extension from the first amplification oligomer to producean eighth amplicon comprising cU2, cD1_(p), and cD2, (iv) the eighthamplicon serves as a template for extension from the seventhamplification oligomer, wherein extension of D2_(p) from a D2_(p):cD2hybrid produces a ninth amplicon comprising U2, D1, and D2_(p), and (v)the ninth amplicon serves as a template for extension from the first orfifth amplification oligomer to produce a tenth amplicon comprising cU2,cD1, and cD2_(p). In a further variation, the target nucleic acid isalso contacted with (8) an eighth amplification oligomer comprising (a)priming segment D2_(p) and (b) displacer tag D3 located 5′ to D2_(p),and (9) a ninth amplification oligomer comprising a third displacerpriming segment D3_(p) having a nucleotide sequence substantiallycorresponding to D3. In such embodiments, the contacting typicallyincludes conditions whereby (i) at least one of the eighth and tenthamplicons serves as a template for extension from the eighthamplification oligomer, wherein extension of D2_(p) from a D2_(p):cD2 orD2_(p):cD2_(p) hybrid produces an eleventh amplicon comprising U2, D1(or D1_(p)), D2_(p), and D3, (ii) the eleventh amplicon serves as atemplate for extension from the first or fifth amplification oligomer toproduce a twelfth amplicon comprising cU2, cD1 (or cD1_(p)), cD2_(p),and cD3, (iii) the twelfth amplicon serves as a template for extensionfrom the ninth amplification oligomer, wherein extension of D3_(p) froma D3_(p):cD3 hybrid produces an thirteenth amplicon comprising U2, D1,D2, and D3_(p), and (iv) the thirteenth amplicon serves as a templatefor extension from the first or fifth amplification oligomer to producea fourteenth amplicon comprising cU2, cD1, cD2, and cD3_(p).

In any of the above embodiments using a T2-U1-D1 reverse amplificationoligomer, the first amplification oligomer may further comprise a fourthheterologous displacer tag (D4) located 5′ to T1 and, optionally, afourth intervening spacer segment (S4) between T1 and D4, whereby thefirst amplicon comprises T1 and D4; and whereby the second ampliconcomprises segments cT1 and cD4, complementary to T1 and D4,respectively. In such variations, the target nucleic acid is furthercontacted with (10) a tenth amplification oligomer comprising a primingsegment T1_(p) having a nucleotide sequence substantially correspondingto T1, or substantially corresponding to the complement of a secondamplicon target sequence cT1′ near or overlapping with cT1 and situated5′ to cD4, and (11) an eleventh amplification oligomer comprising afourth displacer priming segment D4_(p) having a nucleotide sequencesubstantially corresponding to D4, under conditions whereby the secondamplicon serves as a template for extension from both the tenth andeleventh amplification oligomers, wherein extension of T1_(p) from aT1_(p):cT1 or T1_(p):cT1′ hybrid produces a fifteenth amplicon, andwherein extension of D4_(p) from a D4_(p):cD4 hybrid produces asixteenth amplicon while displacing the fifteenth amplicon.

In other embodiments using a T2-U1-D1 reverse amplification oligomer asset forth above, the first amplification oligomer comprises U2 andfurther comprises a fourth heterologous displacer tag (D4) located 5′ toU2 and, optionally, a fourth intervening spacer segment (S4) between U2and D4, whereby the first amplicon comprises U2 and D4, and whereby thesecond amplicon comprises segments cU2 and cD4, complementary to U2 andD4, respectively. Typically, in such variations, the target nucleic acidis further contacted with (10) an eleventh amplification oligomercomprising a fourth displacer priming segment D4_(p) having a nucleotidesequence substantially corresponding to D4, under conditions whereby thesecond amplicon serves as a template for extension from both the fifthand tenth amplification oligomers, wherein extension of U2_(p) from aU2_(p):cU2 hybrid produces a fifteenth amplicon, and wherein extensionof D4_(p) from a D4_(p):cD4 hybrid produces a sixteenth amplicon whiledisplacing the fifteenth amplicon.

In certain embodiments of the method using a T2-U1-D1 reverseamplification oligomer as set forth above, the affinity of D1_(p) forits complement is lower than that of U1. In other embodiments comprisingthe use of a D2p displacer oligomer, (a) the affinity of D1_(p) for itscomplement is lower than that of U1 and/or (b) the affinity of D2_(p)for its complement is lower than that of D1_(p). In yet otherembodiments comprising the use of a D3_(p) displacer oligomer, (a) theaffinity of D1_(p) for its complement is lower than that of U1 and/or(b) the affinity of D2_(p) for its complement is lower than that ofD1_(p) and/or (c) the affinity of D3_(p) for its complement is lowerthan that of D2_(p).

In some embodiments of the method using a T2-U1-D1 reverse amplificationoligomer as set forth above, the target nucleic acid is RNA. In somesuch embodiments, extension from the first amplification oligomercomprises contacting the target nucleic acid with a reversetranscriptase (RT). In more particular variations, the firstamplification oligomer further comprises an RNA polymerase promotersequence (P) (e.g., a T7 promoter sequence) located 5′ to T1, such thatthe second amplicon comprises a segment cP, complementary to thepromoter sequence; and contacting the target nucleic acid furtherincludes conditions whereby an RNA polymerase initiates transcriptionupon recognizing a double-stranded promoter sequence (P:cP) formed byextension of the second amplification oligomer on the first amplicon,thereby producing an RNA amplicon.

In more particular embodiments of the above method utilizing either aT1-U1-D1 forward amplification oligomer or a T2-U1-D1 reverseamplification oligomer, U1 and D1 are different (i.e., have differentnucleotide sequences); in alternative embodiments, U1 and D1 are thesame. In more particular embodiments of the above method utilizingeither a T1-U1-D1 forward amplification oligomer or a T2-U1-D1 reverseamplification oligomer and further comprising a D2 displacer tag, atleast two (e.g., all three) of U1, D1, and D2 are different; inalternative embodiments, at least two (e.g., all three) of U1, D1, andD2 are the same. In yet other embodiments further comprising a D3displacer tag, at least two (e.g., three or all four) of U1, D1, D2, andD3 are different; in alternative embodiments, at least two (e.g., threeor all four) of U1, D1, D2, and D3 are the same.

In other aspects, the present invention provides a kit or reactionmixture for amplifying nucleic acid target region. In some embodiments,the kit or reaction mixture includes (1) a first amplification oligomercomprising (a) a target-binding priming segment (T1) substantiallycomplementary to a 3′-end of the target region, (b) a first heterologousdisplacer tag (D1) located 5′ to T1, and (c) optionally, an interveningspacer segment (S1) between T1 and D1; (2) a second amplificationoligomer comprising a second priming segment T2 substantiallycomplementary to the complement of a 5′-end of the target region; (3) athird amplification oligomer comprising target-binding priming segmentT1_(p) having a nucleotide sequence substantially corresponding to T1,or substantially corresponding to the complement of a target sequencethat is within the target region and near or overlapping with the T1target sequence; and (4) a fourth amplification oligomer comprising adisplacer priming segment D1_(p) having a nucleotide sequencesubstantially corresponding to D1. In some such embodiments, the firstamplification oligomer further comprises a second heterologous displacertag (D2) located 5′ to D1 and, optionally, a second intervening spacersegment (S2) between D1 and D2; alternatively or additionally, the kitor reaction mixture further includes a fifth amplification oligomercomprising (a) priming segment D1_(p) and (b) displacer tag D2 located5′ to D1_(p). In some such variations, the kit or reaction mixturefurther includes a sixth amplification oligomer comprising a seconddisplacer priming segment D2_(p) having a nucleotide sequencesubstantially corresponding to D2. In other embodiments of the above kitor reaction mixture, the first amplification oligomer further comprisesa third heterologous displacer tag (D3) located 5′ to D2 and,optionally, a third intervening spacer segment (S3) between D2 and D3.In some variations, the kit or reaction mixture further includes aseventh amplification oligomer comprising (a) priming segment D2_(p) and(b) displacer tag D3 located 5′ to D2_(p). In more particular variationsof a kit or reaction mixture comprising a D3 tag, the kit or reactionmixture further includes an eighth amplification oligomer comprising athird displacer priming segment D3_(p) having a nucleotide sequencesubstantially corresponding to D3. In some embodiments of a kit orreaction mixture as above, the second amplification oligomer furtherincludes a fourth heterologous displacer tag (D4) located 5′ to T2 and,optionally, a fourth intervening spacer segment (S4) between T2 and D4,and the kit or reaction mixture further includes a ninth amplificationoligomer comprising a priming segment T2_(p) having a nucleotidesequence substantially corresponding to T2 or substantiallycorresponding to a target sequence that is within the complement of thetarget region and near or overlapping with the T2 target sequence, and atenth amplification oligomer comprising a fourth displacer primingsegment D4_(p) having a nucleotide sequence substantially correspondingto D4. In certain variations, the kit or reaction mixture furtherincludes a reverse transcriptase (RT); in some such variations, the kitor reaction mixture further includes an RNA polymerase (e.g., a T7polymerase), wherein the second amplification oligomer further comprisesan RNA polymerase promoter sequence located 5′ to T2.

In other embodiments, a kit or reaction mixture for amplifying a nucleicacid target region includes (1) a first amplification oligomercomprising a target-binding priming segment (T1) substantiallycomplementary to a 3′-end of the target region; (2) a secondamplification oligomer comprising (a) a target-binding segment T2substantially complementary to the complement of a 5′-end of the targetregion, (b) a first heterologous displacer tag (D1) located 5′ to T2,and (c) optionally, an intervening spacer segment (S1) between T2 andD1; (3) a third amplification oligomer comprising target-binding primingsegment T2_(p) having a nucleotide sequence substantially correspondingto T2, or substantially corresponding to a target sequence that iswithin the complement of the target region and near or overlapping withthe T2 target sequence; and (4) a fourth amplification oligomercomprising a displacer priming segment D1_(p) having a nucleotidesequence substantially corresponding to D1. In some such embodiments,the second amplification oligomer further comprises a secondheterologous displacer tag (D2) located 5′ to D1 and, optionally, asecond intervening spacer segment (S2) between D1 and D2; alternativelyor additionally, the kit or reaction mixture further includes a fifthamplification oligomer comprising (a) priming segment D1_(p) and (b)displacer tag D2 located 5′ to D1_(p). In some such variations, the kitor reaction mixture further includes a sixth amplification oligomercomprising a second displacer priming segment D2_(p) having a nucleotidesequence substantially corresponding to D2. In other embodiments of theabove kit or reaction mixture, the second amplification oligomer furthercomprises a third heterologous displacer tag (D3) located 5′ to D2 and,optionally, a third intervening spacer segment (S3) between D2 and D3.In some variations, the kit or reaction mixture further includes aseventh amplification oligomer comprising (a) priming segment D2_(p) and(b) displacer tag D3 located 5′ to D2_(p). In more particular variationsof a kit or reaction mixture comprising a D3 tag, the kit or reactionmixture further includes an eighth amplification oligomer comprising athird displacer priming segment D3_(p) having a nucleotide sequencesubstantially corresponding to D3. In some embodiments of a kit orreaction mixture as above, the first amplification oligomer furtherincludes a fourth heterologous displacer tag (D4) located 5′ to T1 and,optionally, a fourth intervening spacer segment (S4) between T1 and D4,and the kit or reaction mixture further includes a ninth amplificationoligomer comprising a priming segment T1_(p) having a nucleotidesequence substantially corresponding to T1 or substantiallycorresponding to the complement of a target sequence that is within thetarget region and near or overlapping with the T1 target sequence, and atenth amplification oligomer comprising a fourth displacer primingsegment D4_(p) having a nucleotide sequence substantially correspondingto D4. In certain variations, the kit or reaction mixture furtherincludes a reverse transcriptase (RT); in some such variations, the kitor reaction mixture further includes an RNA polymerase (e.g., a T7polymerase), wherein the first amplification oligomer further comprisesan RNA polymerase promoter sequence located 5′ to T1.

In yet other embodiments, a kit or reaction mixture for amplifying anucleic acid target region includes (1) a first amplification oligomercomprising (a) a target-binding priming segment (T1) substantiallycomplementary to a 3′-end of the target region, (b) a heterologousuniversal tag (U1) located 5′ to T1, (c) a first heterologous displacertag (D1) located 5′ to U1, and (d) optionally, an intervening spacersegment (S1) between U1 and D1; (2) a second amplification oligomercomprising (a) a target-binding segment T2 substantially complementaryto the complement of a 5′-end of the target region and (b) optionally, aheterologous universal tag (U2) located 5′ to T2; (3) a thirdamplification oligomer comprising a universal priming segment U1_(p)having a nucleotide sequence substantially corresponding to U1; (4) afourth amplification oligomer comprising a displacer priming segmentD1_(p) having a nucleotide sequence substantially corresponding to D1;and (5) if the second amplification oligomer comprises U2, a fifthamplification oligomer comprising a second universal priming segmentU2_(p) having a nucleotide sequence substantially corresponding to U2.In some such embodiments, the first amplification oligomer furthercomprises a second heterologous displacer tag (D2) located 5′ to D1 and,optionally, a second intervening spacer segment (S2) between D1 and D2;alternatively or additionally, the kit or reaction mixture furtherincludes a sixth amplification oligomer comprising (a) priming segmentD1_(p) and (b) displacer tag D2 located 5′ to D1_(r). In some suchvariations, the kit or reaction mixture further includes a seventhamplification oligomer comprising a second displacer priming segmentD2_(p) having a nucleotide sequence substantially corresponding to D2.In other embodiments of the above kit or reaction mixture, the firstamplification oligomer further comprises a third heterologous displacertag (D3) located 5′ to D2 and, optionally, a third intervening spacersegment (S3) between D2 and D3. In some variations, the kit or reactionmixture further includes an eighth amplification oligomer comprising (a)priming segment D2_(p) and (b) displacer tag D3 located 5′ to D2_(p). Inmore particular variations of a kit or reaction mixture comprising a D3tag, the kit or reaction mixture further includes an eighthamplification oligomer comprising a third displacer priming segmentD3_(p) having a nucleotide sequence substantially corresponding to D3.In some embodiments of a kit or reaction mixture as above, the secondamplification oligomer further comprises a fourth heterologous displacertag (D4) located 5′ to T2 and, optionally, a fourth intervening spacersegment (S4) between T2 and D4, and the kit or reaction mixture furtherincludes a tenth amplification oligomer comprising a priming segmentT2_(p) having a nucleotide sequence substantially corresponding to T2 orsubstantially corresponding to a target sequence that is within thecomplement of the target region and near or overlapping with the T2target sequence, and an eleventh amplification oligomer comprising afourth displacer priming segment D4_(p) having a nucleotide sequencesubstantially corresponding to D4. In other embodiments, the secondamplification oligomer comprises U2 and further comprises a fourthheterologous displacer tag (D4) located 5′ to U2 and, optionally, afourth intervening spacer segment (S4) between U2 and D4, and the kit orreaction mixture further includes a tenth amplification oligomercomprising a fourth displacer priming segment D4_(p) having a nucleotidesequence substantially corresponding to D4. In certain variations, thekit or reaction mixture further includes a reverse transcriptase (RT);in some such variations, the kit or reaction mixture further includes anRNA polymerase (e.g., a T7 polymerase), wherein the second amplificationoligomer further comprises an RNA polymerase promoter sequence located5′ to T2.

In still other embodiments, a kit or reaction mixture for amplifying anucleic acid target region includes (1) a first amplification oligomercomprising (a) a target-binding priming segment (T1) substantiallycomplementary to a 3′-end of the target nucleic acid and (b) optionally,a heterologous universal tag (U2) located 5′ to T1; (2) a secondamplification oligomer comprising (a) a target-binding segment T2substantially complementary to the complement of a 5′-end of the targetregion, (b) a heterologous universal tag (U1) located 5′ to T2, (c) afirst heterologous displacer tag (D1) located 5′ to U1, and (d),optionally, an intervening spacer segment between U1 and D1; (3) a thirdamplification oligomer comprising a universal priming segment U1_(p)having a nucleotide sequence substantially corresponding to U1; (4) afourth amplification oligomer comprising a displacer priming segmentD1_(p) having a nucleotide sequence substantially corresponding to D1;and (5) if the first amplification oligomer comprises U2, a fifthamplification oligomer comprising a second universal priming segmentU2_(p) having a nucleotide sequence substantially corresponding to U2.In some such embodiments, the second amplification oligomer furthercomprises a second heterologous displacer tag (D2) located 5′ to D1 and,optionally, a second intervening spacer segment (S2) between D1 and D2;alternatively or additionally, the kit or reaction mixture furtherincludes a sixth amplification oligomer comprising (a) priming segmentD1_(p) and (b) displacer tag D2 located 5′ to D1,. In some suchvariations, the kit or reaction mixture further includes a seventhamplification oligomer comprising a second displacer priming segmentD2_(p) having a nucleotide sequence substantially corresponding to D2.In other embodiments of the kit or reaction mixture, the secondamplification oligomer further comprises a third heterologous displacertag (D3) located 5′ to D2 and, optionally, a third intervening spacersegment (S3) between D2 and D3. In some variations, the kit or reactionmixture further includes an eighth amplification oligomer comprising (a)priming segment D2_(p) and (b) displacer tag D3 located 5′ to D2_(p). Inmore particular variations of a kit or reaction mixture comprising a D3tag, the kit or reaction mixture further includes an eighthamplification oligomer comprising a third displacer priming segmentD3_(p) having a nucleotide sequence substantially corresponding to D3.In some embodiments of a kit or reaction mixture as above, the firstamplification oligomer further comprises a fourth heterologous displacertag (D4) located 5′ to T1 and, optionally, a fourth intervening spacersegment (S4) between T1 and D4, and the kit or reaction mixture furtherincludes a tenth amplification oligomer comprising a priming segmentT1_(p) having a nucleotide sequence substantially corresponding to T1 orsubstantially corresponding to the complement of a target sequence thatis within the target region and near or overlapping with the T1 targetsequence, and an eleventh amplification oligomer comprising a fourthdisplacer priming segment D4_(p) having a nucleotide sequencesubstantially corresponding to D4. In other embodiments, the firstamplification oligomer comprises U2 and further comprises a fourthheterologous displacer tag (D4) located 5′ to U2 and, optionally, afourth intervening spacer segment (S4) between U2 and D4, and the kit orreaction mixture further includes a tenth amplification oligomercomprising a fourth displacer priming segment D4_(p) having a nucleotidesequence substantially corresponding to D4. In certain variations, thekit or reaction mixture further includes a reverse transcriptase (RT);in some such variations, the kit or reaction mixture further includes anRNA polymerase (e.g., a T7 polymerase), wherein the first amplificationoligomer further comprises an RNA polymerase promoter sequence located5′ to T1.

These and other aspects of the invention will become evident uponreference to the following detailed description of the invention and theattached drawings.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art pertinent to the methods and compositions described. As usedherein, the following terms and phrases have the meanings ascribed tothem unless specified otherwise.

The terms “a,” “an,” and “the” include plural referents, unless thecontext clearly indicates otherwise. For example, “a nucleic acid” asused herein is understood to represent one or more nucleic acids. Assuch, the terms “a” (or “an”), “one or more,” and “at least one” can beused interchangeably herein.

The term “nucleic acid” is intended to encompass a singular “nucleicacid” as well as plural “nucleic acids,” and refers to any chain of twoor more nucleotides, nucleosides, or nucleobases (e.g.,deoxyribonucleotides or ribonucleotides) covalently bonded together.Nucleic acids include, but are not limited to, viral genomes, orportions thereof, either DNA or RNA, bacterial genomes, or portionsthereof, fungal, plant or animal genomes, or portions thereof, messengerRNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), plasmid DNA,mitochondrial DNA, or synthetic DNA or RNA. A nucleic acid may beprovided in a linear (e.g., mRNA), circular (e.g., plasmid), or branchedform, as well as a double stranded or single stranded form. Nucleicacids may include modified bases to alter the function or behavior ofthe nucleic acid, e.g., addition of a 3′-terminal dideoxynucleotide toblock additional nucleotides from being added to the nucleic acid. Asused herein, a “sequence” of a nucleic acid refers to the sequence ofbases which make up a nucleic acid. The term “polynucleotide” may beused herein to denote a nucleic acid chain Throughout this application,nucleic acids are designated as having a 5′-terminus and a 3′-terminus.Standard nucleic acids, e.g., DNA and RNA, are typically synthesized“3′-to-5′,” i.e., by the addition of nucleotides to the 5′-terminus of agrowing nucleic acid.

A “nucleotide” is a subunit of a nucleic acid consisting of a phosphategroup, a 5-carbon sugar and a nitrogenous base. The 5-carbon sugar foundin RNA is ribose. In DNA, the 5-carbon sugar is 2′-deoxyribose. The termalso includes analogs of such subunits, such as a methoxy group at the2′ position of the ribose (2′-O-Me). As used herein, methoxyoligonucleotides containing “T” residues have a methoxy group at the 2′position of the ribose moiety, and a uracil at the base position of thenucleotide.

The term “region,” with reference to a nucleic acid, refers to a portionof the nucleic acid where the portion is smaller than the entire nucleicacid.

A “target nucleic acid” is a nucleic acid comprising a “target region”to be amplified. Target nucleic acids may be DNA or RNA as describedherein, and may be either single-stranded or double-stranded. The targetnucleic acid may include other regions besides the target region whichmay not be amplified. Typical target nucleic acids include viralgenomes, bacterial genomes, fungal genomes, plant genomes, animalgenomes, rRNA, tRNA, or mRNA from viruses, bacteria or eukaryotic cells,mitochondrial DNA, or chromosomal DNA.

Target nucleic acids may be isolated from any number of sources based onthe purpose of the amplification assay being carried out. Sources oftarget nucleic acids include, but are not limited to, clinical specimens(e.g., blood, either whole blood or platelets, urine, saliva, feces,semen, or spinal fluid), environmental samples (e.g., water or soilsamples), food samples, beverages, industrial samples (e.g., productsand process materials, including water), seed stocks, cDNA libraries, ortotal cellular RNA. By “isolated” it is meant that a sample containing atarget nucleic acid is taken from its natural milieu; however, the termdoes not connote any particular degree of purification. If necessary,target nucleic acids of the present invention are made available forinteraction with the various oligonucleotides of the present invention.This may include, for example, cell lysis or cell permeabilization torelease the target nucleic acid from cells, which then may be followedby one or more purification steps, such as a series of isolation andwash steps. (See, e.g., U.S. Pat. Nos. 5,786,208 and 6,821,770, eachincorporated by reference herein.) This may be particularly importantwhere the sample source or cellular material released into the samplecan interfere with the amplification reaction. Methods to prepare targetnucleic acids from various sources for amplification are well known tothose of ordinary skill in the art. Target nucleic acids of the presentinvention may be purified to some degree prior to the amplificationreactions described herein, but in other cases, the sample is added tothe amplification reaction without any further manipulations.

The term “nucleic acid target region” or “target region” refers to aparticular nucleotide sequence of the target nucleic acid that is to beamplified. The “target region” includes the complexing sequences towhich amplification oligomers and any associated detection probe(s)complex during the processes of the present invention. Unless thecontext clearly dictates otherwise, where the target nucleic acid isoriginally single-stranded, the term “target region” will also refer tothe sequence complementary to the target region as present in the targetnucleic acid; and where the target nucleic acid is originallydouble-stranded, the term “target region” refers to both the sense (+)and antisense (−) strands. Notwithstanding the above, reference toeither a 3′- or 5′-end of a target region is understood to refer to aparticular strand of the target region (for example, reference to atarget-binding priming segment in terms of its substantialcomplementarity to a “3′-end of the target region” is understood to meanthat the target-binding segment is substantially complementary to the3′-end a single strand of the target region). The optimal length of atarget sequence depends on a number of considerations, for example, theamount of secondary structure, or self-hybridizing regions in thesequence. A target sequence of the present invention may be of anypractical length. Determining the optimal length is easily accomplishedby those of ordinary skill in the art using routine optimizationmethods. In typical embodiments, a target region ranges from about 100nucleotides in length to from about 150 to about 250 nucleotides inlength. The optimal or preferred length may vary under differentconditions, which can easily be tested by one of ordinary skill in theart.

The term “target sequence” generally refers to a smaller nucleic acidsequence region within a larger nucleic acid sequence (e.g., within anucleic acid target region) that hybridizes specifically to at least aportion of an amplification oligomer or probe oligomer (e.g., detectionprobe) by standard base pairing.

In choosing a target region, and particularly in choosing a targetsequence within the target region, the skilled artisan will understandthat a “unique” sequence should be chosen so as to distinguish betweenunrelated or closely related target nucleic acids. As will be understoodby those of ordinary skill in the art, “unique” sequences are judgedfrom the testing environment. At least the sequences recognized by thetarget-binding segment of an amplification oligonucleotide and anyassociated detection probe (as described in more detail elsewhereherein) should be unique in the environment being tested, but need notbe unique within the universe of all possible sequences. In someembodiments, it may be desirable to choose a target region or targetsequence which is common to a class of organisms, for example, asequence which is common to all E. coli strains that might be in asample. In other situations, a very highly specific target region, or atarget region having at least a highly specific target sequencerecognized by a detection probe, would be chosen so as to distinguishbetween closely related organisms, for example, between pathogenic andnon-pathogenic E. coli.

As used herein, the term “oligonucleotide,” “oligo,” or “oligomer” isintended to encompass a singular “oligonucleotide” as well as plural“oligonucleotides,” and refers to any polymer of two or more ofnucleotides, nucleosides, nucleobases or related compounds used as areagent in the amplification methods of the present invention, as wellas subsequent detection methods. The oligonucleotide may be DNA and/orRNA and/or analogs thereof. The term oligonucleotide does not denote anyparticular function to the reagent; rather, it is used generically tocover all such reagents described herein. An oligonucleotide may servevarious different functions. For example, it may function as a primer ifit is capable of hybridizing to a complementary strand and can furtherbe extended in the presence of a nucleic acid polymerase; it may providea promoter if it contains a sequence recognized by an RNA polymerase andallows for transcription; and it may function to prevent hybridizationor impede primer extension if appropriately situated and/or modified.Specific oligonucleotides of the present invention are described in moredetail below. As used herein, an oligonucleotide can be virtually anylength, limited only by its specific function in an amplificationreaction or in detecting an amplification product of the amplificationreaction.

Oligonucleotides of a defined sequence and chemical structure may beproduced by techniques known to those of ordinary skill in the art, suchas by chemical or biochemical synthesis, and by in vitro or in vivoexpression from recombinant nucleic acid molecules, e.g., bacterial orviral vectors. As intended by this disclosure, an oligonucleotide doesnot consist solely of wild-type chromosomal DNA or the in vivotranscription products thereof.

Oligonucleotides may be modified in any way, as long as a givenmodification is compatible with the desired function of a givenoligonucleotide. One of ordinary skill in the art can easily determinewhether a given modification is suitable or desired for any givenoligonucleotide of the present invention. Modifications include basemodifications, sugar modifications or backbone modifications. Basemodifications include, but are not limited to the use of the followingbases in addition to adenine, cytidine, guanosine, thymine and uracil:C-5 propyne, 2-amino adenine, 5-methyl cytidine, inosine, and dP and dKbases. The sugar groups of the nucleoside subunits may be ribose,deoxyribose and analogs thereof, including, for example, ribonucleosideshaving a 2′-O-methyl (2′-O-ME) substitution to the ribofuranosyl moiety.(See, e.g., U.S. Pat. No. 6,130,038, incorporated by reference herein.)Other sugar modifications include, but are not limited to 2′-amino,2′-fluoro, (L)-alpha-threofuranosyl, and pentopuranosyl modifications.The nucleoside subunits may be joined by linkages such as phosphodiesterlinkages, modified linkages or by non-nucleotide moieties which do notprevent hybridization of the oligonucleotide to its complementary targetnucleic acid sequence. Modified linkages include those linkages in whicha standard phosphodiester linkage is replaced with a different linkage,such as a phosphorothioate linkage or a methylphosphonate linkage. Thenucleobase subunits may be joined, for example, by replacing the naturaldeoxyribose phosphate backbone of DNA with a pseudo peptide backbone,such as a 2-aminoethylglycine backbone which couples the nucleobasesubunits by means of a carboxymethyl linker to the central secondaryamine (DNA analogs having a pseudo peptide backbone are commonlyreferred to as “peptide nucleic acids” or “PNA” and are disclosed inU.S. Pat. No. 5,539,082, incorporated by reference herein.) Otherlinkage modifications include, but are not limited to, morpholino bonds.

Non-limiting examples of oligonucleotides or oligomers contemplated bythe present invention include nucleic acid analogs containing bicyclicand tricyclic nucleoside and nucleotide analogs (LNAs). (See, e.g., U.S.Pat. Nos. 6,268,490 and 6,670,461, each incorporated by referenceherein.) Any nucleic acid analog is contemplated by the presentinvention provided the modified oligonucleotide can perform its intendedfunction, e.g., hybridize to a target nucleic acid under stringenthybridization conditions or amplification conditions, or interact with aDNA or RNA polymerase, thereby initiating extension or transcription. Inthe case of detection probes, the modified oligonucleotides must also becapable of preferentially hybridizing to the target nucleic acid understringent hybridization conditions.

While design and sequence of oligonucleotides for the present inventiondepend on their function as described below, several variables mustgenerally be taken into account. Among the most critical are: length,melting temperature (T_(m)), specificity, complementarity with otheroligonucleotides in the system, G/C content, polypyrimidine (T, C) orpolypurine (A, G) stretches, and the 3′-end sequence. Controlling forthese and other variables is a standard and well-known aspect ofoligonucleotide design, and various computer programs are readilyavailable to screen large numbers of potential oligonucleotides foroptimal ones.

“Substantially complementary” means that a nucleic acid strand (e.g., atarget-binding sequence of an amplification oligomer) is capable ofhybridizing to a target nucleic acid strand (e.g., to a target sequencewithin a nucleic acid target region). “Hybridization” means sufficienthydrogen bonding, which can be, e.g., Watson-Crick, Hoogsteen, orreversed Hoogsteen hydrogen bonding, between complementary nucleoside ornucleotide bases such that stable and specific binding occurs betweenthe nucleic acid strands. Hybridization capability is determinedaccording to amplification conditions or stringent hybridizationconditions, including suitable buffer concentrations and temperatures,that allow specific hybridization to a nucleic acid strand having aregion of full or partial complementarity. Thus, not all nucleotides ofthe nucleic acid need by complementary. Further, a nucleic acid strandis “substantially complementary” when it hybridizes to all, part, or anoverlapping region of a target nucleic acid. Qualitative andquantitative considerations for establishing amplification conditions orstringent hybridization conditions for the design of oligonucleotidesaccording to the present invention are known in the art. Typically, twonucleic acid regions are substantially complementary when, e.g., atleast 90% of the respective bases are complementary, more typically whenat least 95% and preferably when 100% of the respective bases arecomplementary.

A nucleotide sequence of an oligonucleotide “substantially correspondsto” a specified reference nucleic acid sequence if the nucleotidesequence is sufficiently similar to the reference sequence such that theoligonucleotide has similar hybridization properties to the referencenucleic acid sequence in that it would hybridize with the same targetnucleic acid sequence under the conditions employed (amplificationconditions or stringent hybridization conditions). One skilled in theart will understand that “substantially corresponding oligonucleotides”can vary from a reference sequence and still hybridize to the sametarget nucleic acid sequence. It is also understood that a first nucleicacid corresponding to a second nucleic acid includes the RNA and DNAthereof and includes the complements thereof, unless the context clearlydictates otherwise. In certain variations of a nucleotide sequencedescribed herein as “substantially corresponding” to a specifiedreference sequence, the nucleotide sequence is identical to thereference sequence.

The 3′-terminus of an oligonucleotide (or other nucleic acid) can beblocked in a variety of ways using a blocking moiety, as describedbelow. A “blocked” oligonucleotide is not efficiently extended by theaddition of nucleotides to its 3′-terminus, by a DNA- or RNA-dependentDNA polymerase, to produce a complementary strand of DNA. As such, a“blocked” oligonucleotide cannot be a “primer.”

The term “segment,” as used herein with reference to an oligonucleotide(e.g., an amplification oligomer), refers to a discrete polynucleotidethat may form all or only a part of the oligonucleotide and which istypically associated with a specific function. Thus, an oligonucleotidemay contain one more segments as described herein. For example, anamplification oligomer may include a “target-binding segment,” which iscapable of hybridizing to a target sequence, and a “displacer tagsegment,” which provides a displacer priming sequence. In anotherexample, an amplification oligomer may comprise a “priming segment” forpriming polymerase-mediated nucleotide extension, and such an oligomermay or may not include additional nucleotides in addition to the primingsegment. The term “segment” is also used herein to refer tooligonucleotide segments or their complements that have beenincorporated into an amplicon.

Reference herein to two specified segments separated by a forward slash(“/”) is a reference to the specified segments in the alternative. Thus,for example, reference to a segment as “D1/D1_(p)” means that thespecified segment is either D1 or D1_(p), depending on the particularcontext.

A “tagged oligonucleotide” as used herein refers to an oligonucleotidethat comprises at least a first region and a second region, where thefirst region is a “target-binding segment” that hybridizes to the 3′-endof a nucleic acid target region of interest, and where the second regionis a “tag segment” situated 5′ to the target-binding segment and whichdoes not stably hybridize or bind to a target nucleic acid containingthe target region. In accordance with the present invention, thetarget-binding segment of a tagged oligonucleotide is typically apriming sequence. Thus, the tagged oligonucleotide is typically a“tagged priming oligonucleotide” comprising a tag segment and atarget-binding priming segment. In certain aspects, the taggedoligonucleotide is a “tagged promoter oligonucleotide” comprising a tagsegment, a target-binding priming segment, and a promoter sequence (or“promoter segment”) situated 5′ to the tag segment and effective forinitiating transcription therefrom. The features and designconsiderations for a target-binding priming segment are the same as forpriming oligonucleotides.

The “tag segment” (also referred to herein as “tag,” “heterologous tag,”or “heterologous tag segment”) may have essentially any heterologousnucleotide sequence provided that it does not stably hybridize to thetarget nucleic acid sequence of interest and, thereby, participate indetectable amplification. The heterologous tag preferably does notstably hybridize to any sequence derived from the genome of an organismbeing tested or, more particularly, to any target nucleic acid underreaction conditions. A tag segment that is present in a taggedoligonucleotide is preferably designed so as not to substantially impairor interfere with the ability of the target-binding segment to hybridizeto its target sequence. Moreover, the tag segment will be of sufficientlength and composition such that once a complement of the tag has beenincorporated into an initial DNA primer extension product, atag-specific priming oligonucleotide can then be used to participate insubsequent rounds of amplification as described herein. A tag segment ofthe present invention is typically at least 10 nucleotides in length,and may extend up to 15, 20, 25, 30, 35, 40, 50 or more nucleotides inlength. Skilled artisans will recognize that the design of tag sequencesand tagged oligonucleotides for use in the present invention can followany of a number of suitable strategies, while still achieving theobjectives and advantages described herein.

By “amplification” or “nucleic acid amplification” is meant productionof multiple copies of a nucleic acid target region. The multiple copiesmay be referred to as “amplicons” or “amplification products.” Anamplicon produced by polymerase-mediated extension from an amplificationprimer (i.e., an amplification oligomer containing at least a 3′ primingsegment), when hybridized to a target nucleic acid template, is also bereferred to herein as an “extension product.” In certain embodiments,the amplified target region contains less than the complete target genesequence (introns and exons) or an expressed target gene sequence(spliced transcript of exons and flanking untranslated sequences). Forexample, specific amplicons may be produced by amplifying a portion ofthe target nucleic acid by using amplification primers that hybridizeto, and initiate polymerization from, internal positions of the targetnucleic acid. Preferably, the amplified portion contains a detectabletarget sequence that may be detected using any of a variety ofwell-known methods.

Many well-known methods of nucleic acid amplification requirethermocycling to alternately denature double-stranded nucleic acids andhybridize primers; however, other well-known methods of nucleic acidamplification are isothermal. The polymerase chain reaction (see Mulliset al., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159, eachincorporated by reference herein), commonly referred to as PCR, usesmultiple cycles of denaturation, annealing of primer pairs to oppositestrands, and primer extension to exponentially increase copy numbers ofthe target sequence. In a variation called RT-PCR, reverse transcriptase(RT) is used to make a complementary DNA (cDNA) from mRNA, and the cDNAis then amplified by PCR to produce multiple copies of DNA (see Gelfandet al., U.S. Pat. Nos. 5,322,770 and 5,310,652, each incorporated byreference herein). Another method is strand displacement amplification,commonly referred to as SDA, which uses cycles of annealing pairs ofprimer sequences to opposite strands of a target sequence, primerextension in the presence of a dNTP to produce a duplexhemiphosphorothioated primer extension product, endonuclease-mediatednicking of a hemimodified restriction endonuclease recognition site, andpolymerase-mediated primer extension from the 3′-end of the nick todisplace an existing strand and produce a strand for the next round ofprimer annealing, nicking and strand displacement, resulting ingeometric amplification of product. (See Walker et al., Proc. Natl.Acad. Sci. USA 89:392-396, 1992; Walker et al., Nucleic Acids Research20:1691-1696, 1992; and U.S. Pat. No. 5,455,166; each incorporated byreference herein.) Thermophilic SDA (tSDA) uses thermophilicendonucleases and polymerases at higher temperatures in essentially thesame method (see European Pat. No. 0 684 315, incorporated by referenceherein). Other amplification methods include: nucleic acid sequencebased amplification (see Malek et al., U.S. Pat. No. 5,130,238,incorporated by reference herein), commonly referred to as NASBA; onethat uses an RNA replicase to amplify the probe molecule itself (seeLizardi et al., BioTechnol. 6:1197-1202, 1988, incorporated by referenceherein), commonly referred to as Qβ replicase; a transcription-basedamplification method (see Kwoh, et al., Proc. Natl. Acad. Sci. USA86:1173-1177, 1989, incorporated by reference herein); self-sustainedsequence replication (see Guatelli et al., Proc. Natl. Acad. Sci. USA87, 1874-1878, 1990; Landgren, Trends in Genetics 9:199-202, 1993; andLee et al., NUCLEIC ACID AMPLIFICATION TECHNOLOGIES (1997); eachincorporated by reference herein); and, transcription-mediatedamplification (see Kacian et al., U.S. Pat. Nos. 5,480,784 and5,399,491, each incorporated by reference herein), commonly referred toas TMA. For further discussion of known amplification methods seePersing, David H., “In Vitro Nucleic Acid Amplification Techniques” inDiagnostic Medical Microbiology: Principles and Applications (Persing etal. eds., 1993), pp. 51-87, incorporated by reference herein. Otherillustrative amplification methods suitable for use in accordance withthe present invention include rolling circle amplification (RCA) (seeLizardi, U.S. Pat. No. 5,854,033, incorporated by reference herein);Helicase Dependent Amplification (HDA) (see Kong et al., U.S. Pat.Appln. Pub. No. US 2004-0058378 A1, incorporated by reference herein);and Loop-Mediated Isothermal Amplification (LAMP) (see Notomi et al.,U.S. Pat. No. 6,410,278, incorporated by reference herein).

Preferred transcription-based amplification systems of the presentinvention include TMA, which employs an RNA polymerase to producemultiple RNA transcripts of a target region (see, e.g., Kacian et al.,U.S. Pat. Nos. 5,480,784 and 5,399,491; and Becker et al., U.S. Pat. No.7,374,885; each incorporated by reference herein). TMA uses a “promoteroligonucleotide” or “promoter-primer” that hybridizes to a targetnucleic acid in the presence of a reverse transcriptase and an RNApolymerase to form a double-stranded promoter from which the RNApolymerase produces RNA transcripts. These transcripts can becometemplates for further rounds of TMA in the presence of a second primercapable of hybridizing to the RNA transcripts. Unlike PCR, LCR or othermethods that require heat denaturation, TMA is an isothermal method thatuses an RNAse H activity to digest the RNA strand of an RNA:DNA hybrid,thereby making the DNA strand available for hybridization with a primeror promoter-primer. Typically, the RNAse H activity associated with thereverse transcriptase provided for amplification is used.

In one illustrative TMA method, one amplification primer is anoligonucleotide promoter-primer that comprises a promoter sequence,which becomes functional when double-stranded, located 5′ of atarget-binding sequence that is capable of hybridizing to a binding siteof a target RNA at a location 3′ to the sequence to be amplified. Apromoter-primer may be referred to as a “T7-primer” when it is specificfor T7 RNA polymerase recognition. Under certain circumstances, the3′-end of a promoter-primer, or a subpopulation of suchpromoter-primers, may be modified to block or reduce primer extension.From an unmodified promoter-primer, reverse transcriptase creates a cDNAcopy of the target RNA, while RNAse H activity degrades the target RNA.A second amplification primer then binds to the cDNA. This primer may bereferred to as a “non-T7 primer” to distinguish it from a “T7-primer.”From this second amplification primer, reverse transcriptase createsanother DNA strand, resulting in a double-stranded DNA with a functionalpromoter at one end. When double-stranded, the promoter sequence iscapable of binding an RNA polymerase to begin transcription of thetarget sequence to which the promoter-primer is hybridized. An RNApolymerase uses this promoter sequence to produce multiple RNAtranscripts (i.e., amplicons), generally about 100 to 1,000 copies. Eachnewly-synthesized amplicon can anneal with the second amplificationprimer. Reverse transcriptase can then create a DNA copy, while theRNAse H activity degrades the RNA of this RNA:DNA duplex. Thepromoter-primer can then bind to the newly synthesized DNA, allowing thereverse transcriptase to create a double-stranded DNA, from which theRNA polymerase produces multiple amplicons. Thus, a billion-foldisothermic amplification can be achieved using two amplificationprimers.

In another illustrative TMA method, one or more features as described inBecker et al., U.S. Pat. Appln. Pub. No. US 2006-0046265 A1, areoptionally incorporated. Preferred TMA methods in this respect includethe use of blocking moieties, terminating moieties, and other modifyingmoieties that provide improved TMA process sensitivity and accuracy.Thus, certain preferred embodiments of the present invention employtagged oligonucleotides and tag-mediated displacement, as describedherein, in conjunction with the methods as described in Becker et al.,U.S. Pat. Appln. Pub. No. US 2006-0046265 A1.

By “detectable amplification” is meant that a detectable signalassociated with an amplification product in an amplification reactionmixture rises above a predetermined background or threshold level(end-point amplification) or rises above a background or threshold levelwithin a predetermined period of time (real-time amplification). (See,e.g., Light et al., U.S. Pat. Appln. Pub. No. US 2006-0276972,paragraphs 506-549, incorporated by reference herein.) The amplificationproduct contains a sequence having sequence identity with a nucleic acidtarget region or its complement and can be detected with, for example,an intercalating dye or a detection probe having specificity for atarget sequence within the target region or its complement.

“Amplification conditions” means conditions permitting nucleic acidamplification according to the present invention. Amplificationconditions may, in some embodiments, be less stringent than “stringenthybridization conditions” as described herein. Oligonucleotides used inthe amplification reactions of the present invention hybridize to theirintended targets under amplification conditions, but may or may nothybridize under stringent hybridization conditions. On the other hand,detection probes of the present invention hybridize under stringenthybridization conditions. While the Examples section infra providesexemplary amplification conditions for amplifying target nucleic acidsequences according to the present invention, other acceptableconditions to carry out nucleic acid amplifications according to thepresent invention could be easily ascertained by someone having ordinaryskill in the art depending on the particular method of amplificationemployed.

“Nucleic acid hybridization” is the process by which two nucleic acidstrands having completely or partially complementary nucleotidesequences come together under predetermined reaction conditions to forma stable, double-stranded hybrid. Either nucleic acid strand may be adeoxyribonucleic acid (DNA) or a ribonucleic acid (RNA) or analogsthereof. Thus, hybridization can involve RNA:RNA hybrids, DNA:DNAhybrids, RNA:DNA hybrids, or analogs thereof. The two constituentstrands of this double-stranded structure, sometimes called a hybrid,are held together by hydrogen bonds. Although these hydrogen bonds mostcommonly form between nucleotides containing the bases adenine andthymine or uracil (A and T or U) or cytosine and guanine (C and G) onsingle nucleic acid strands, base pairing can also form between baseswhich are not members of these “canonical” pairs. Non-canonical basepairing is well-known in the art. (See, e.g., Roger L. P. Adams et al.,The Biochemistry of the Nucleic Acids (11th ed. 1992), incorporated byreference herein.)

“Stringent hybridization conditions” or “stringent conditions” refer toconditions where a specific detection probe is able to hybridize withtarget nucleic acids over other nucleic acids present in the testsample. It will be appreciated that these conditions may vary dependingupon factors including the GC content and length of the probe, thehybridization temperature, the composition of the hybridization reagentor solution, and the degree of hybridization specificity sought.Stringent hybridization conditions can include, for example,6×NaCl/sodium citrate (SSC) at about 45° C. for a hybridization step,followed by a wash of 2×SSC at 50° C.; or, alternatively, e.g.,hybridization at 42° C. in 5×SSC, 20 mM NaPO4, pH 6.8, 50% formamide,followed by a wash of 0.2×SSC at 42° C.

By “nucleic acid hybrid” or “hybrid” or “duplex” is meant a nucleic acidstructure containing a double-stranded, hydrogen-bonded region whereeach strand is substantially complementary to the other, and where theregion is sufficiently stable under amplification conditions orstringent hybridization conditions. Such hybrids may comprise RNA:RNA,RNA:DNA, or DNA:DNA duplex molecules.

By “stable” or “stably hybridize” is meant that the temperature of areaction mixture is at least 2° C. below the melting temperature of anucleic acid duplex.

“Target-binding sequence” or “target-binding segment” is used herein torefer to a nucleic acid sequence that is substantially complementary to,and thus configured to hybridize with, a target nucleic acid sequence.

An “amplification oligonucleotide” is an oligonucleotide, at least the3′-end of which is substantially complementary to a target nucleic acid,and which hybridizes to a target nucleic acid, or its complement, andparticipates in a nucleic acid amplification reaction. An example of anamplification oligonucleotide is a “primer” or “priming oligonucleotide”that hybridizes to a target nucleic acid and contains a 3′ OH end thatis extended by a polymerase in an amplification process. Another exampleof an amplification oligonucleotide is an oligonucleotide that is notextended by a polymerase (e.g., because it has a 3′ blocked end) butparticipates in or facilitates amplification. In some embodiments, the5′ region of an amplification oligonucleotide includes a promotersequence that is non-complementary to the target nucleic acid (which maybe referred to as a “promoter primer” or “promoter provider”). Thoseskilled in the art will understand that an amplification oligonucleotidethat functions as a primer may be modified to include a 5′ promotersequence, and thus function as a “promoter primer.” Incorporating a 3′blocked end further modifies the promoter primer, which is now capableof hybridizing to a target nucleic acid and providing an upstreampromoter sequence that serves to initiate transcription, but does notprovide a primer for oligo extension. Such a modified oligo is referredto herein as a “promoter provider” oligonucleotide. Size ranges foramplification oligonucleotides include those that are about 10 to about70 nt long (not including any promoter sequence or poly-A tails) andcontain at least about 10 contiguous bases, or even at least 12contiguous bases that are complementary to a region of the targetnucleic acid sequence (or a complementary strand thereof). Anamplification oligonucleotide may optionally include modifiednucleotides or analogs, or additional nucleotides that participate in anamplification reaction but are not complementary to or contained in thetarget nucleic acid, or template sequence.

As is well known in the art, a “promoter” is a specific nucleic acidsequence that is recognized by a DNA-dependent RNA polymerase(“transcriptase”) as a signal to bind to the nucleic acid and begin thetranscription of RNA at a specific site. For binding, it was generallythought that such transcriptases required DNA which had been rendereddouble-stranded in the region comprising the promoter sequence via anextension reaction. It is now known that efficient transcription of RNAcan take place even under conditions where a double-stranded promoter isnot formed through an extension reaction with the template nucleic acid.The template nucleic acid (the sequence to be transcribed) need not bedouble-stranded. Individual DNA-dependent RNA polymerases recognize avariety of different promoter sequences, which can vary markedly intheir efficiency in promoting transcription. When an RNA polymerasebinds to a promoter sequence to initiate transcription, that promotersequence is not part of the sequence transcribed. Thus, the RNAtranscripts produced thereby will not include that sequence.

A “promoter provider” is an amplification oligonucleotide comprisingfirst and second regions, and which is preferably modified to preventthe initiation of DNA synthesis from its 3′-terminus. The first regionof a promoter oligonucleotide is a “target-binding segment” having abase sequence that hybridizes to a DNA template, where thetarget-binding segment is situated 3′, but not necessarily adjacent to,a promoter region. The hybridizing portion of a promoter oligonucleotideis typically at least 10 nucleotides in length, and may extend up to 15,20, 25, 30, 35, 40, 50 or more nucleotides in length. The second regionis a “promoter segment” comprising a promoter for an RNA polymerase. Apromoter provider oligonucleotide is engineered so that it is incapableof being extended by an RNA- or DNA-dependent DNA polymerase, e.g.,reverse transcriptase, preferably comprising a blocking moiety at its3′-terminus as described herein.

A “priming oligonucleotide” is an oligonucleotide, at least the 3′-endof which is complementary to a nucleic acid template, and whichcomplexes (by hydrogen bonding or hybridization) with the template togive a primer:template complex suitable for initiation of synthesis byan RNA- or DNA-dependent DNA polymerase. The at least 3′-end of thepriming oligonucleotide that is complementary to the nucleic acidtemplate is also referred to herein a “priming sequence” or “primingsegment” and in certain variations a priming oligonucleotide may includeadditional nucleotide(s) 5′ to the priming sequence that do not stablyhybridize with the target nucleic acid. A priming oligonucleotide isextended by the addition of covalently bonded nucleotide bases to its3′-terminus, which bases are complementary to the template. The resultis a primer extension product. A priming oligonucleotide of the presentinvention is typically at least 10 nucleotides in length, and may extendup to 15, 20, 25, 30, 35, 40, 50 or more nucleotides in length. Suitableand preferred priming oligonucleotides are described herein. Virtuallyall DNA polymerases (including reverse transcriptases) that are knownrequire complexing of an oligonucleotide to a single-stranded template(“priming”) to initiate DNA synthesis, whereas RNA replication andtranscription (copying of RNA from DNA) generally do not require aprimer. By its very nature of being extended by a DNA polymerase, apriming oligonucleotide does not comprise a 3′-blocking moiety.

A “displacer oligonucleotide” is a priming oligonucleotide comprising a“displacer priming sequence” (also referred to herein as a “displacerpriming segment”) that hybridizes to a template nucleic acid upstreamfrom a neighboring priming oligonucleotide hybridized to the 3′-end of atarget sequence (referred to herein as the “forward primingoligonucleotide”). By “upstream” is meant that a 3′-end of the displaceroligonucleotide complexes with the template nucleic acid 5′ to a 3′-endof the forward priming oligonucleotide. When hybridized to the templatenucleic acid, the 3′-terminal base of the displacer oligonucleotide ispreferably adjacent to or spaced from the 5-terminal base of the forwardpriming oligonucleotide. More preferably, the 3′-terminal base of thedisplacer oligonucleotide is spaced from 5 to 35 bases from the5′-terminal base of the forward priming oligonucleotide. The displaceroligonucleotide may be provided to a reaction mixture contemporaneouslywith the forward priming oligonucleotide or after the forward primingoligonucleotide has had sufficient time to hybridize to the templatenucleic acid. Extension of the forward priming oligonucleotide can beinitiated prior to or after the displacer oligonucleotide is provided toa reaction mixture. Under amplification conditions, the displaceroligonucleotide is extended in a template-dependent manner, therebydisplacing a primer extension product comprising the forward primingoligonucleotide that is complexed with the template nucleic acid. Oncedisplaced from the template nucleic acid, the primer extension productcomprising the forward priming oligonucleotide is available forcomplexing with another amplification oligomer. The forward primingoligonucleotide and the displacer oligonucleotide both preferentiallyhybridize to the target nucleic acid. Examples of displaceroligonucleotides and their uses are disclosed by Becker et al., U.S.Pat. No. 7,713,697, incorporated by reference herein.

A “displacer tag” or “heterologous displacer tag” as used herein refersto a tag segment having a nucleotide sequence that substantiallycorresponds to a displacer priming sequence of a displaceroligonucleotide, such that once a complement of the displacer tag hasbeen incorporated into an oligonucleotide primer extension product, thedisplacer oligonucleotide can then be used to participate in subsequentrounds of amplification as described herein. Typically, the displacertag sequence is the same as the displacer priming sequence.

A “universal priming oligonucleotide” is a priming oligonucleotidecomprising a “universal priming sequence” (also referred to herein as a“universal priming segment”) that does not specifically hybridize to anucleic acid target region, but instead hybridizes to a sequence that isthe complement of a heterologous tag (a “universal tag”), such that oncea complement of the universal tag has been incorporated into anoligonucleotide primer extension product, the universal primingoligonucleotide can then be used to participate in subsequent rounds ofamplification as described herein. Accordingly, a “universal tag” or“heterologous universal tag” as used herein refers to a tag segmenthaving a nucleotide sequence that substantially corresponds to auniversal priming sequence of a universal priming oligonucleotide.Typically, the universal tag sequence is the same as the universalpriming sequence. A universal tag is typically situated 3′ to adisplacer tag.

As used herein, a “spacer segment” or “intervening spacer segment” is anucleotide sequence positioned between and adjacent to two othersegments of an oligonucleotide and which is not a target-bindingsequence, a sequence substantially corresponding to a priming sequence,or a promoter sequence. Spacer segments are typically 1 to 20nucleotides in length, more typically 3 to 18 nucleotides in length, andmost typically 3 to 12 nucleotides in length, including all wholenumbers therebetween. In some aspects, the inclusion of spacer segmentsin amplification oligomers comprising a displacer tag (e.g., between atarget-binding segment and a displacer tag) can improve displacementactivity. In other aspects, the inclusion of spacer segments in promoteroligonucleotides (e.g., between a target-binding segment and an RNApolymerase promoter sequence) increases the rate at which RNAamplification products are formed.

As used herein, a “blocking moiety” is a substance used to “block” the3′-terminus of an oligonucleotide or other nucleic acid so that itcannot be efficiently extended by a nucleic acid polymerase. A blockingmoiety may be a small molecule, e.g., a phosphate or ammonium group, orit may be a modified nucleotide, e.g., a 3′2′ dideoxynucleotide or 3′deoxyadenosine 5′-triphosphate (cordycepin), or other modifiednucleotide. Additional blocking moieties include, for example, the useof a nucleotide or a short nucleotide sequence having a 3′-to-5′orientation, so that there is no free hydroxyl group at the 3′-terminus,the use of a 3′ alkyl group, a 3′ non-nucleotide moiety (see, e.g.,Arnold et al., U.S. Pat. No. 6,031,091, incorporated by referenceherein), phosphorothioate, alkane-diol residues, peptide nucleic acid(PNA), nucleotide residues lacking a 3′ hydroxyl group at the3′-terminus, or a nucleic acid binding protein. Preferably, the3′-blocking moiety comprises a nucleotide or a nucleotide sequencehaving a 3′-to-5′ orientation or a 3′ non-nucleotide moiety, and not a3′2′-dideoxynucleotide or a 3′ terminus having a free hydroxyl group.Additional methods to prepare 3′-blocking oligonucleotides arewell-known to those of ordinary skill in the art.

A “terminating oligonucleotide” is an oligonucleotide comprising a basesequence that is complementary to a region of the target nucleic acid inthe vicinity of the 5′-end of the target sequence, so as to “terminate”primer extension of a nascent nucleic acid that includes a primingoligonucleotide, thereby providing a defined 3′-end for the nascentnucleic acid strand. A terminating oligonucleotide is designed tohybridize to the target nucleic acid at a position sufficient to achievethe desired 3′-end for the nascent nucleic acid strand. The positioningof the terminating oligonucleotide is flexible depending upon itsdesign. A terminating oligonucleotide may be modified or unmodified. Incertain embodiments, terminating oligonucleotides are synthesized withat least one or more 2′-O-ME ribonucleotides. These modified nucleotideshave demonstrated higher thermal stability of complementary duplexes.The 2′-O-ME ribonucleotides also function to increase the resistance ofoligonucleotides to exonucleases, thereby increasing the half-life ofthe modified oligonucleotides. (See, e.g., Majlessi et al., NucleicAcids Res. 26:2224-9, 1988, incorporated by reference herein.) Othermodifications as described elsewhere herein may be utilized in additionto or in place of 2′-O-ME ribonucleotides. For example, a terminatingoligonucleotide may comprise PNA or an LNA. (See, e.g., Petersen et al.,J. Mol. Recognit. 13:44-53, 2000, incorporated by reference herein.) Aterminating oligonucleotide of the present invention typically includesa blocking moiety at its 3′-terminus to prevent extension. A terminatingoligonucleotide may also comprise a protein or peptide joined to theoligonucleotide so as to terminate further extension of a nascentnucleic acid chain by a polymerase. A terminating oligonucleotide of thepresent invention is typically at least 10 bases in length, and mayextend up to 15, 20, 25, 30, 35, 40, 50 or more nucleotides in length.Suitable and preferred terminating oligonucleotides are describedherein. While a terminating oligonucleotide typically or necessarilyincludes a 3′-blocking moiety, “3′-blocked” oligonucleotides are notnecessarily terminating oligonucleotides. Other oligonucleotides of thepresent invention, e.g., promoter oligonucleotides and cappingoligonucleotides are typically or necessarily 3′-blocked as well.

“Detection probe” refers to an oligonucleotide comprising atarget-binding segment that hybridizes specifically to a target sequencecontained within a nucleic acid target region under conditions thatpromote hybridization to allow detection of a target nucleic acidcomprising the target region, or an amplified nucleic acid comprisingthe target region. Detection may either be direct (e.g., a probehybridized directly to its target sequence) or indirect (e.g., a probelinked to its target via an intermediate molecular structure). Detectionprobes may be DNA, RNA, analogs thereof or combinations thereof and theymay be labeled or unlabeled. Detection probes may further includealternative backbone linkages such as, e.g., 2′-O-methyl linkages. Adetection probe may comprise target-specific sequences and othersequences that contribute to the three-dimensional conformation of theprobe (see, e.g., U.S. Pat. Nos. 5,118,801; 5,312,728; 6,849,412;6,835,542; 6,534,274; and 6,361,945; and US Patent Application Pub. No.20060068417; each incorporated by reference herein).

As used herein, a “label” refers to a moiety or compound joined directlyor indirectly to a probe that is detected or leads to a detectablesignal. Direct labeling can occur through bonds or interactions thatlink the label to the probe, including covalent bonds or non-covalentinteractions, e.g., hydrogen bonds, hydrophobic and ionic interactions,or formation of chelates or coordination complexes. Indirect labelingcan occur through use of a bridging moiety or “linker” such as a bindingpair member, an antibody or additional oligomer, which is eitherdirectly or indirectly labeled, and which may amplify the detectablesignal. Labels include any detectable moiety, such as a radionuclide,ligand (e.g., biotin, avidin), enzyme or enzyme substrate, reactivegroup, or chromophore (e.g., dye, particle, or bead that impartsdetectable color), luminescent compound (e.g., bioluminescent,phosphorescent, or chemiluminescent labels), or fluorophore. Labels maybe detectable in a homogeneous assay in which bound labeled probe in amixture exhibits a detectable change different from that of an unboundlabeled probe, e.g., instability or differential degradation properties.A “homogeneous detectable label” can be detected without physicallyremoving bound from unbound forms of the label or labeled probe (see,e.g., U.S. Pat. Nos. 5,283,174; 5,656,207; and 5,658,737; eachincorporated by reference herein). Labels include chemiluminescentcompounds, e.g., acridinium ester (“AE”) compounds that include standardAE and derivatives (see, e.g., U.S. Pat. Nos. 5,656,207; 5,658,737; and5,639,604; each incorporated by reference herein). Synthesis and methodsof attaching labels to nucleic acids and detecting labels are wellknown. (See, e.g., Sambrook et al., Molecular Cloning, A LaboratoryManual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Habor,N Y, 1989), Chapter 10, incorporated by reference herein. See also U.S.Pat. Nos. 5,658,737; 5,656,207; 5,547,842; 5,283,174; and 4,581,333;each incorporated by reference herein). More than one label, and morethan one type of label, may be present on a particular probe, ordetection may use a mixture of probes in which each probe is labeledwith a compound that produces a detectable signal (see, e.g., U.S. Pat.Nos. 6,180,340 and 6,350,579, each incorporated by reference herein).

“Capture probe” or “capture oligomer” refers to an oligonucleotidecomprising a target-binding segment that hybridizes specifically to atarget sequence in a target nucleic acid and a moiety that joins to abinding partner on an immobilized probe to capture the target nucleicacid to a support. One example of a capture probe includes two bindingregions: the target-binding segment and an immobilized probe-bindingregion, usually on the same oligomer, although the two regions may bepresent on two different oligomers joined together by one or morelinkers. Another embodiment of a capture oligomer uses a target-bindingsegment that includes random or non-random poly-GU, poly-GT, or poly Usequences to bind non-specifically to a target nucleic acid and link itto an immobilized probe on a support.

“Immobilized oligonucleotide” or “immobilized probe” refers to a nucleicacid binding partner that joins a capture oligomer to a support,directly or indirectly. An immobilized probe joined to a supportfacilitates separation of a capture probe bound target from unboundmaterial in a sample. One embodiment of an immobilized probe is anoligonucleotide joined to a support that facilitates separation of boundtarget nucleic acid from unbound material in a sample. Supports mayinclude known materials, such as matrices and particles free insolution, which may be made of nitrocellulose, nylon, glass,polyacrylate, mixed polymers, polystyrene, silane, polypropylene, metal,or other compositions, of which one embodiment is magneticallyattractable particles. Supports may be monodisperse magnetic spheres(e.g., uniform size ±5%), to which an immobilized probe is joineddirectly (via covalent linkage, chelation, or ionic interaction), orindirectly (via one or more linkers), where the linkage or interactionbetween the probe and support is stable during hybridization conditions.

“Sample preparation” refers to any steps or method that treats a samplefor subsequent amplification and/or detection of target nucleic acidspresent in the sample. Samples may be complex mixtures of components ofwhich the target nucleic acid is a minority component. Samplepreparation may include any known method of concentrating components,such as microbes or nucleic acids, from a larger sample volume, such asby filtration of airborne or waterborne particles from a larger volumesample or by isolation of microbes from a sample by using standardmicrobiology methods. Sample preparation may include physical disruptionand/or chemical lysis of cellular components to release intracellularcomponents into a substantially aqueous or organic phase and removal ofdebris, such as by using filtration, centrifugation or adsorption.Sample preparation may include use of a nucleic acid oligonucleotidethat selectively or non-specifically capture a target nucleic acid andseparate it from other sample components (e.g., as described in U.S.Pat. No. 6,110,678 and International Patent Application Pub. No. WO2008/016988, each incorporated by reference herein).

“Separating” or “purifying” means that one or more components of asample are removed or separated from other sample components. Samplecomponents include target nucleic acids usually in a generally aqueoussolution phase, which may also include cellular fragments, proteins,carbohydrates, lipids, and other nucleic acids. Separating or purifyingremoves at least 70%, or at least 80%, or at least 95% of the targetnucleic acid from other sample components.

The term “specificity,” in the context of an amplification and/ordetection system, is used herein to refer to the characteristic of thesystem which describes its ability to distinguish between target andnon-target nucleic acids dependent on sequence and assay conditions. Interms of nucleic acid amplification, specificity generally refers to theratio of the number of specific amplicons produced to the number ofside-products (e.g., the signal-to-noise ratio). In terms of detection,specificity generally refers to the ratio of signal produced from targetnucleic acids to signal produced from non-target nucleic acids.

The term “sensitivity” is used herein to refer to the precision withwhich a nucleic acid amplification reaction can be detected orquantitated. The sensitivity of an amplification reaction is generally ameasure of the smallest copy number of the target nucleic acid that canbe reliably detected in the amplification system, and will depend, forexample, on the detection assay being employed, and the specificity ofthe amplification reaction, e.g., the ratio of specific amplicons toside-products.

As used herein, the term “relative light unit” (“RLU”) is an arbitraryunit of measurement indicating the relative number of photons emitted bythe sample at a given wavelength or band of wavelengths. RLU varies withthe characteristics of the detection means used for the measurement.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict the steps of a tag-mediated displacementamplification reaction utilizing a first heterologous displacer tag.TNA: initial target nucleic acid or non-primer, amplicon portion thereofin + polarity; T1: target-binding priming segment; D1: firstheterologous displacer tag segment; S1: first intervening spacersegment; tna: non-primer, amplicon portion of target nucleic acid in −polarity; T2: priming segment substantially corresponding to 2ndamplicon; c[X]: amplicon regions complementary to segment [X]; T1_(p):priming segment substantially corresponding to T1; D1_(p): primingsegment substantially corresponding to D1; AP[#]: amplification product.

FIGS. 2A and 2B depict the steps of a tag-mediated displacementamplification reaction utilizing a first heterologous displacer tagtogether with a heterologous universal tag. TNA: initial target nucleicacid or non-primer, amplicon portion thereof in + polarity; T1:target-binding priming segment; U1: heterologous universal tag segment;D1: first heterologous displacer tag segment; S1: first interveningspacer segment; tna: non-primer, amplicon portion of target nucleic acidin − polarity; T2: priming segment substantially corresponding to 2ndamplicon; c[X]: amplicon regions complementary to segment [X]; U1_(p):priming segment substantially corresponding to U1; D1_(p): primingsegment substantially corresponding to D1; AP[#]: amplification product.

FIGS. 3A and 3B depict the use of a second heterologous displacer tag inthe amplification reaction of FIGS. 1A and 1B. TNA: initial targetnucleic acid or non-primer, amplicon portion thereof in + polarity; T1:target-binding priming segment; D1: first heterologous displacer tagsegment; S1: first intervening spacer segment; D2: second heterologousdisplacer segment; S2: second intervening spacer segment; tna:non-primer, amplicon portion of target nucleic acid in − polarity; T2:priming segment substantially corresponding to 2nd amplicon; c[X]:amplicon regions complementary to segment [X]; T1_(p): priming segmentsubstantially corresponding to T1; D1_(p): priming segment substantiallycorresponding to D1; D2_(p): priming segment substantially correspondingto D1; AO1: first amplification oligonucleotide; AP[#]: amplificationproduct.

FIG. 4 depicts the use of an additional displacer oligonucleotidecomprising a first displacer priming segment and a second heterologousdisplacer tag in the amplification reaction of FIGS. 3A and 3B. TNA:initial target nucleic acid or non-primer, amplicon portion thereof in +polarity; T1: target-binding priming segment; D1: first heterologousdisplacer tag segment; S1: first intervening spacer segment; D2: secondheterologous displacer segment; S2: second intervening spacer segment;tna: non-primer, amplicon portion of target nucleic acid in − polarity;T2: priming segment substantially corresponding to 2nd amplicon; c[X]:amplicon regions complementary to segment [X]; D1_(p): priming segmentsubstantially corresponding to D1; D2_(p): priming segment substantiallycorresponding to D1; AP[#]: amplification product.

FIG. 5 depicts the use of a third heterologous displacer tag in theamplification reaction of FIGS. 1A and 1B. TNA: initial target nucleicacid or non-primer, amplicon portion thereof in + polarity; T1:target-binding priming segment; D1: first heterologous displacer tagsegment; S1: first intervening spacer segment; D2: second heterologousdisplacer segment; S2: second intervening spacer segment; D3: thirdheterologous displacer segment; S3: third interventing spacer segment;tna: non-primer, amplicon portion of target nucleic acid in − polarity;T2: priming segment substantially corresponding to 2nd amplicon; c[X]:amplicon regions complementary to segment [X]; T1_(p): priming segmentsubstantially corresponding to T1; D1_(p): priming segment substantiallycorresponding to D1; D2_(p): priming segment substantially correspondingto D1; D3_(p): priming segment substantially corresponding to D1; AO1:first amplification oligonucleotide; AP[#]: amplification product.

FIG. 6 depicts the use of an additional displacer oligonucleotidecomprising a second displacer priming segment and a third heterologousdisplacer tag in the amplification reaction of FIGS. 3A and 3B. TNA:initial target nucleic acid or non-primer, amplicon portion thereof in +polarity; T1: target-binding priming segment; D1: first heterologousdisplacer tag segment; S1: first intervening spacer segment; D2: secondheterologous displacer segment; S2: second intervening spacer segment;D3: third heterologous displacer segment; S3: third interventing spacersegment; tna: non-primer, amplicon portion of target nucleic acid in −polarity; T2: priming segment substantially corresponding to 2ndamplicon; c[X]: amplicon regions complementary to segment [X]; D1_(p):priming segment substantially corresponding to D1; D2_(p): primingsegment substantially corresponding to D1; AP[#]: amplification product.

FIG. 7 depicts the use of a reverse priming oligonucleotide comprising adisplacer tag in the amplification reaction of FIGS. 1A and 1B. TNA:initial target nucleic acid or non-primer, amplicon portion thereof in +polarity; T1: target-binding priming segment; D1: first heterologousdisplacer tag segment; S1: first intervening spacer segment; D4: fourthheterologous displacer tag segment; S4: fourth intervening spacersegment; tna: non-primer, amplicon portion of target nucleic acid in −polarity; T2: priming segment substantially corresponding to 2ndamplicon; c[X]: amplicon regions complementary to segment [X]; T1_(p):priming segment substantially corresponding to T1; D1_(p): primingsegment substantially corresponding to D1; D4_(p): priming segmentsubstantially corresponding to D4; AO2: second amplificationoligonucleotide; AP[#]: amplification product.

FIG. 8 depicts the use of a promoter primer in the amplificationreaction of FIGS. 1A and 1B. TNA: initial target nucleic acid ornon-primer, amplicon portion thereof in + polarity; T1: target-bindingpriming segment; D1: first heterologous displacer tag segment; S1: firstintervening spacer segment; tna: non-primer, amplicon portion of targetnucleic acid in − polarity; T2: priming segment substantiallycorresponding to 2nd amplicon; P: promoter segment; c[X]: ampliconregions complementary to segment [X]; D1_(p): priming segmentsubstantially corresponding to D1; AO2: second amplificationoligonucleotide; AP[#]: amplification product; RNA POL: RNA polymerase.

FIG. 9 depicts the use of a promoter provider in a tag-mediateddisplacement amplification reaction utilizing a first heterologousdisplacer tag. TNA: initial target nucleic acid or non-primer, ampliconportion thereof in + polarity; T1: target-binding priming segment; D1:first heterologous displacer tag segment; S1: first intervening spacersegment; tna: non-primer, amplicon portion of target nucleic acid in −polarity; TERM: terminating oligonucleotide; T2: target-binding segmentsubstantially corresponding to 2nd amplicon; P: promoter segment; X:blocking moiety; c[X]: amplicon regions complementary to segment [X];T1_(p): priming segment substantially corresponding to T1; AP[#]:amplification product; RNA POL: RNA polymerase.

FIG. 10 depicts the amplification capacity of a tag-mediateddisplacement amplification reaction utilizing a universal priming siteand a displacer priming site incorporated into an amplicon, togetherwith three amplification oligomers: a universal priming oligomer, adisplacer priming oligomer comprising a displacer priming segment; and asecond displacer priming oligomer comprising the displacer primingsegment and a heterologous displacer tag. TNA: initial target nucleicacid or non-primer, amplicon portion thereof in + polarity; T1:target-binding priming segment; U1: heterologous universal tag segment;D1: first heterologous displacer tag segment; S1: first interveningspacer segment; D2: second heterologous displacer segment; S2: secondintervening spacer segment; tna: non-primer, amplicon portion of targetnucleic acid in − polarity; T2: priming segment substantiallycorresponding to 2nd amplicon; c[X]: amplicon regions complementary tosegment [X]; U1_(p): priming segment substantially corresponding to U1;D1_(p): priming segment substantially corresponding to D1; D2_(p):priming segment substantially corresponding to D1; AP[#]: amplificationproduct.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods, kits, and reaction mixtures foramplification of nucleic acids using a tag-mediated displacementstrategy. An amplification oligonucleotide is equipped with aheterologous displacer tag situated 5′ to a target-binding segment andhaving a sequence that substantially corresponds to the priming sequenceof a displacer oligonucleotide. In this manner, once the complement ofthe displacer tag has been incorporated into an amplification product,thereby providing a displacer priming site, the displaceroligonucleotide can participate in subsequent rounds of amplificationfor displacement of an extension product primed from a site within theamplification product 5′ to the displacer priming site, therebyincreasing overall amplification out put and increasing assay kineticsand sensitivity.

This strategy can be used to improve the kinetics of amplificationreactions as well as amplification capacity. For example, anamplification oligomer can be designed to include one or more additionaldisplacer tags, each situated in succession 5′ to the initial displacertag. Additional displacer oligomers may then be included in the ampreaction, each corresponding to at least one additional tag and designedto bind an amplicon at a site 3′ to at least one other displaceroligomer. Using multiple displacer tags and oligomers in this manner,additional amplification products can be efficiently produced in eachround of the amp reaction, thereby increasing overall amplificationoutput and increasing assay kinetics and sensitivity.

Tag-mediated displacement as described herein is also advantageous, forexample, when using heterologous tags to introduce universal primingsites. A non-target-specific “universal” priming site can beincorporated into an amplification product using an amplificationoligomer equipped with a universal tag substantially corresponding tothe universal tag and located 5′ to an initial target-specific primingsegment. Once a complement of the universal tag has been incorporatedinto an amplification product, a universal priming oligonucleotide canbe used to participate in subsequent rounds of amplification. Such ascenario would preclude the use of known displacement methods, whichrely on the use of target-specific displacer priming sites. Using,however, a tag-mediated displacement strategy, a heterologous displacertag can be incorporated into an amplification oligomer at a site 5′ tothe universal tag, such that the amplification product comprising theuniversal priming site further comprises a 3′ displacer priming site,thereby allowing the use of a displacer oligomer in subsequentamplification. Not only will extension of a universal primer from theuniversal priming site be displaced, but extension of the displaceroligomer will produce an additional amplification product, therebyincreasing overall amplification output and increasing assay kineticsand sensitivity.

The present invention can be adapted for use in essentially anyamplification procedure requiring a template-binding primingoligonucleotide capable of extension in the presence of a nucleic acidpolymerase. Incorporation of heterologous displacer tags into suchamplification procedures can be achieved without substantially modifyingthe reagents and reaction conditions of such procedures. Any neededmodifications would be well within the knowledge and capabilities of askilled molecular biologist in view of the instant description.Descriptions of various amplification procedures adopting tag-mediateddisplacement are further provided herein.

FIGS. 1A and 1B illustrate one embodiment of the nucleic acidamplification method utilizing tag-mediated displacement. Step 1 shows afirst amplification oligonucleotide comprising a target-binding primingsegment (T1) and a heterologous displacer tag (D1) located 5′ to T1. Thefirst amplification oligonucleotide further includes an optionalintervening spacer segment (S1) between T1 and D1. The target-bindingpriming segment hybridizes to a target nucleic acid (TNA) at a 3′-end ofa target region. An extension reaction is initiated from the 3′-end ofthe hybridized, tagged oligonucleotide with a DNA polymerase to producea first amplicon comprising the displacer tag D1 and a regioncomplementary to the target nucleic acid (tna). See FIG. 1A, Steps 1 and2. The first amplicon is separated from the target nucleic acid (seeStep 3), and a second amplification oligonucleotide comprising atarget-binding segment T2, substantially complementary to the complementof a 5′-end of the target region, hybridizes to the first extensionproduct (see Step 4). An amplification reaction (e.g., an extensionreaction, as indicated in the Figure) is initiated from the secondamplification oligomer to produce a second amplicon comprising segmentscT1 and cD1, respectively complementary to T1 and D1. See FIG. 1A, Steps4 to 6. A third amplification oligomer comprising priming segmentT1_(p), having a sequence substantially corresponding to T1, thenhybridizes to the second amplicon to form a T1_(p):cT1 duplex. See id,Steps 6 and 7. In addition, a fourth oligonucleotide primer comprising adisplacer priming segment D1_(p), having a sequence substantiallycorresponding to D1, hybridizes to the second amplicon upstream fromT1_(p) to form a D1_(p):cD1 duplex, and extension reactions areinitiated from both T1_(p) and D1_(p). See id., Step 7. Extension of thethird amplification oligomer produces a third amplicon, which isdisplaced from the second amplicon as the fourth amplification oligomeris extended. See FIG. 1B, Step 8. Full extension of the fourthamplification oligomer produces a fourth amplicon and results incomplete separation of the third amplicon from its template, therebymaking the third amplicon available for further rounds of amplification.See id., Step 9.

FIGS. 2A and 2B illustrate an embodiment utilizing a universal tag,together with the displacer tag, to incorporate a universal priming sitein an amplification product. In this embodiment, the first amplificationoligomer from FIG. 1A further includes a heterologous universal tag (U1)situated 5′ to the target-binding priming segment T1 and 3′ to both theheterologous displacer tag D1 and optional spacer segment S1. See FIG.2A, Step 1. Steps 1 to 4 parallel those shown in FIG. 1A, whereby afirst amplicon is initiated from priming segment T1 hybridized to thetarget nucleic acid, and a second amplicon is initiated from a secondamplification oligomer T2 hybridized to the first amplicon.Amplification from the second oligomer produces a second ampliconcomprising segments cU1 and cD1, respectively complementary to U1 andD1. See id, Step 5 and 6. A third amplification oligomer comprisinguniversal priming segment U1_(p), having a sequence substantiallycorresponding to U1, then hybridizes to the second amplicon to form aU1_(p):cU1 duplex. See id, Step 7. In addition, a fourth oligonucleotideprimer comprising displacer priming segment D1_(p) hybridizes to thesecond extension product upstream from U1_(p) to form a U1_(p):cU1duplex, and extension reactions are initiated from both U1_(p) andU1_(p). See id., Step 7. Steps 8 and 9 (FIG. 2B) illustrate adisplacement reaction parallel to that shown in FIG. 1B. Specifically,extension of the third amplification oligomer produces a third amplicon,which is displaced from the second amplicon as the fourth amplificationoligomer is extended. See FIG. 2B, Step 8. Full extension of the fourthamplification oligomer produces a fourth amplicon and results incomplete separation of the third amplicon from its template, therebymaking the third amplicon available for further rounds of amplification.See id, Step 9. As also illustrated in this figure, once the universaltag and its complement, cU1, are incorporated into the amplificationproducts, amplification can utilize cU1 as a universal priming siteinstead of a target-specific priming site. Any variation of the methodsdescribed can be similarly modified to incorporate the use of universalpriming sites and primers following initial amplification of a targetnucleic acid.

In certain embodiments of the method, multiple displacer tags areincorporated in a tagged oligonucleotide. For example, FIGS. 3A and 3Billustrate an embodiment in which the first amplification oligomer fromFIG. 1A further includes a second heterologous displacer tag D2 situated5′ to D1, with an optional second spacer segment S2 situated between D1and D2. See FIG. 3A, First Amp Oligo. Using this modified first oligomerin Steps 1 through 6 of FIG. 1A results in a second amplicon furthercomprising segment cD2, complementary to D2, located 3′ to cD1. See FIG.3A, EP2. Referring to Step 1 of FIG. 3A, in addition to hybridization ofthe third and fourth amplification oligomers (comprising T1_(p) andD1_(p), respectively) to the second amplicon, a fifth amplificationoligomer comprising a second displacer priming segment D2_(p), having asequence substantially corresponding to D2, hybridizes to the secondamplicon upstream from D1_(p) to form a D2_(p):cD2 duplex, and extensionreactions are initiated from each of T1_(p), D1_(p), and D2_(p). Seeid., Step 1. Extension of the third amplification oligomer from T1_(p)produces a third amplicon, which is displaced from the second amplicontemplate as the fourth amplification oligomer is extended from D1_(p).See FIG. 3A, Step 2. Similarly, the fourth amplicon, produced byextension of the fourth amplification oligomer, is displaced from thetemplate as the fifth amplification oligomer is extended from D2_(p).See id. Production of the fourth amplicon results in complete separationof the third amplicon from the template strand, while full extension ofthe fifth amplification oligomer produces a fifth amplicon, resulting incomplete separation of the fourth amplicon from its template, therebymaking both the third and fourth amplicons available for further roundsof amplification. See id., Step 3. The fifth amplicon then serves as atemplate for amplification from the second amplification oligomercomprising segment T2, thereby producing a sixth amplicon comprisingsegments cT1, cD1 and cD2_(p) (see Steps 5 and 6), which can serve as atemplate for further amplification (e.g., using one or more of oligomerscomprising T1_(p), D1_(p), or D2_(p)).

In other embodiments, an additional displacer oligonucleotide comprisinga second heterologous displacer tag is added to the reaction to furtherincrease amplification capacity. For example, in some variations, anamplification oligonucleotide comprising displacer priming segmentD1_(p) and displacer tag D2, situated 5′ to D1_(p), may be added to anamplification reaction as depicted in FIGS. 3A and 3B. Such an oligomeris particularly useful, e.g., for further amplification of an ampliconcomprising displacer priming site cD1 (or cD1_(p)) while alsoincorporating a second displacer priming site corresponding to D2. Forexample, referring to FIG. 4, AP7 represents a seventh ampliconcomprising cD1_(p), but not cD2, which can be produced by amplificationfrom oligomer T2 hybridized to the fourth amplicon (AP4) of FIG. 3A. Anamplification oligomer comprising priming segment D1_(p), an optionalspacer segment S2, and displacer tag D2 hybridizes to the seventhextension product to form a D1_(p):cD1_(p) duplex, and extension of theamplification oligomer produces an eighth amplicon. See FIG. 4, Steps 1and 2. The eighth extension product then serves as a template foramplification from the second amplification oligomer, comprising segmentT2, to produce a ninth amplicon comprising cD2 in addition to cT1 andcD1_(p). See id., Steps 3 and 4. This ninth amplicon may then be used asa template for, e.g., extension of a D2_(p) displacer priming segment(in addition or alternatively to extension from T1_(p) and/or D1_(p)).

In certain variations, an additional displacer oligonucleotidecomprising displacer priming segment D1_(p) and a second displacer tagD2, as described above, is used in conjunction with an initialamplification oligomer comprising priming segment T1 and both displacertags D1 and D2. In such variations, the D1_(p)-D2 oligomer may increasethe generation of amplicons comprising the D2 tag. For example, asindicated above, the D1_(p)-D2 can prime from amplicons comprising acD1_(p) priming site but lacking cD2 (in addition to the capability ofpriming from amplicons comprising both cD1/cD1_(p) and cD2).

Alternatively, a displacer oligomer comprising D1_(p) and D2 is usedtogether with an initial amplification oligomer that includes primingsegment T1 and displacer tag D1 in the absence of D2. In suchvariations, the D1_(p)-D2 oligomer is also useful for the initialincorporation of the D2 tag into an amplification product. For example,the D1_(p)-D2 oligomer may utilize an amplicon such as that shown inStep 6 of FIG. 1A (AP2) as a template for extension from a D1_(p):cD1duplex to produce an amplicon incorporating the D2 displacer tag. Thisamplicon may then serve as a template for primer extension to produce acomplementary amplicon incorporating a cD2 displacer priming site.

In variations of the method utilizing multiple displacer tags, three ormore displacer tags are employed, together with corresponding displacerpriming oligonucleotide(s), to still further increase amplificationcapacity. FIG. 5 illustrates one such embodiment in which the firstamplification oligomer from FIG. 3A further includes a thirdheterologous displacer tag D3 situated 5′ to D2, with an optional thirdspacer segment S3 situated between D2 and D3. See FIG. 5, First AmpOligo. Using this modified first oligomer in Steps 1 through 6 of FIG.1A results in a second amplicon further comprising segment cD3,complementary to D3, located 3′ to cD2. See FIG. 5, EP2. Referring toStep 1 of FIG. 5, in addition to hybridization of the third, fourth, andfifth amplification oligomers (comprising T1_(p), and D1_(p), andD2_(p), respectively) to the second amplicon, an additionalamplification oligomer comprising a third displacer priming segmentD3_(p), having a sequence substantially corresponding to D3, hybridizesto the second amplicon upstream from D2_(p) to form a D3_(p):cD3 duplex,and extension reactions are initiated from each of T1_(p), D1_(p),D2_(p), and D3_(p). See id., Step 1. Extension of the thirdamplification oligomer from T1_(p) produces a third amplicon, which isdisplaced from the second amplicon template as the fourth amplificationoligomer is extended from D1_(p) to produce a fourth amplicon, which inturn is displaced as the fifth amplification oligomer is extended fromD2_(p) to produce a fifth amplicon, as previously depicted in Step 2 ofFIG. 3A. Here, by inclusion of an additional cD3 priming site and aD3_(p) priming oligomer, the fifth amplicon is similarly displaced fromthe template with extension from D3_(p) to produce a tenth amplicon.Primer extension and displacement thus results in third, fourth, andfifth amplicons completely separated from the template strand, togetherwith the production of an additional extension product, here designatedas a tenth amplicon, AP10. See FIG. 5, Step 2. Upon separation of thetenth amplicon from the template strand, the tenth amplicon serves as atemplate for amplification from the second amplification oligomercomprising segment T2, thereby producing an eleventh amplicon comprisingsegments cT1, cD1, cD2, and cD3_(p) (see Step 3), which can serve as atemplate for further amplification (e.g., using one or more of oligomerscomprising T1_(p), D1_(p), D2_(p), or D3_(p)).

In other embodiments employing the use of three or more displacer tags,a third or further displacer tag(s) may be incorporated into anadditional displacer oligonucleotide in a similar manner previouslydepicted in FIG. 4. In certain variations, for example, an amplificationoligonucleotide comprising displacer priming segment D2_(p) and a thirddisplacer tag D3, situated 5′ to D2_(p), may be added to anamplification reaction as depicted in FIG. 4. Such an oligomer may beused, e.g., for further amplification of an amplicon comprising a seconddisplacer priming site cD2 (or cD2_(p)) while also incorporating a thirddisplacer priming site corresponding to D3. For example, referring toFIG. 6, AP9 represents a ninth amplicon comprising cD2, such as thatproduced by the amplification reaction depicted in FIG. 4. Anamplification oligonucleotide comprising priming segment D2_(p), anoptional spacer segment S3, and displacer tag D3 hybridizes to the ninthamplicon to form a cD2_(p):cD2 duplex. See FIG. 6, Step 1. Extension ofthe oligonucleotide produces a twelfth amplicon, which can bind thesecond amplification oligomer comprising target-binding segment T2 andserve as a template for amplification of a thirteenth ampliconcomprising cD3 in addition to cT1, cD1_(p), and cD2_(p). See id., Step2. This thirteenth amplicon may then be used as a template for, e.g.,extension of a D3_(p) displacer priming segment (in addition oralternatively to extension from T1_(p), D1_(p), and/or D2_(p)).

In various embodiments of the method, the tag-mediated displacementstrategy as described herein may be employed using either a forward orreverse amplification oligonucleotide comprising one or more displacertags. In other embodiments, both a forward and a reverse amplificationoligonucleotide include one or more displacer tags. For example, in theamplification reaction previously depicted in FIGS. 1A and 1B, thesecond amplification oligomer comprising target-binding segment T2 mayalso include a heterologous displacer tag in addition to the firstamplification oligomer. FIG. 7 illustrates such a variation, in whichthe second amplification oligomer comprises displacer tag D4 and anoptional intervening spacer segment situated 5′ to T2. See FIG. 7,Second Amp Oligo. Using this tagged T2 oligonucleotide in the reverseamplification reaction previously depicted in Steps 4 to 6 of FIG. 1Agenerates a second amplicon AP2 comprising D4 as shown in FIG. 7. Primerextension from the D1_(p) displacer oligomer on this AP2 template (aspreviously depicted in Steps 7 to 9 of FIGS. 1A and 1B) then produces afourth amplicon AP4 comprising a cD4 displacer priming site as shown inFIG. 7. Using this fourth amplicon as a template for furtheramplification, a amplification oligomer comprising priming segmentT2_(p), having a sequence substantially corresponding to T2, hybridizesto the second amplicon to form a T2_(p):cT2 duplex, and a displaceroligonucleotide primer comprising a displacer priming segment D4_(p),having a sequence substantially corresponding to D4, hybridizes to thefourth amplicon upstream from T2 to form a D4_(p):cD4 duplex, andextension reactions are initiated from both T2_(p) and D4_(p). See FIG.7, Step 1. Extension of the T2_(p) amplification oligomer produces anextension product designated here as a fourteenth amplicon AP14, whichis displaced from the AP2 template as the displacer oligomer is extendedto produce an extension product designated here as a fifteenth ampliconAP15. See id., Step 2.

Some preferred variations of the amplification method utilize anisothermal, transcription-based amplification reaction known astranscription-based amplification (TMA), various aspects of which aredisclosed in Becker et al., U.S. Pat. No. 7,374,885. As previouslydiscussed herein, TMA employs an RNA polymerase to produce multiple RNAcopies of a target region. A promoter primer or promoter provideroligonucleotide is utilized to incorporate a promoter sequence for theRNA polymerase. Upon formation of a double-stranded promoter, producedby a primer extension reaction on the initial promoter sequence as atemplate, the RNA polymerase binds to the promoter and produces multipleRNA transcripts, which can become templates for further rounds ofamplification in the presence of a priming oligonucleotide capable ofhybridizing to the RNA transcripts.

Accordingly, particular variations of the present method include the useof a promoter primer in the amplification reaction. For example, in theamplification reaction previously depicted in FIGS. 1A and 1B, thesecond amplification oligomer comprising target-binding segment T2 mayfurther include an RNA polymerase promoter sequence. FIG. 8 illustratessuch a variation, in which the second amplification oligomer is apromoter primer comprising a priming segment T2 and a promoter sequenceP situated 5′ to T2. See FIG. 8, Second Amp Oligo. Using this promoterprimer in the reverse amplification reaction previously depicted inSteps 4 to 6 of FIG. 1A generates a second amplicon AP2 comprising P asshown in FIG. 8. Primer extension from the D1_(p) displacer oligomer onthis AP2 template (as previously depicted in Steps 7 to 9 of FIGS. 1Aand 1B) then produces a fourth amplicon AP4 comprising a segment cPcomplementary to the promoter sequence, thereby forming adouble-stranded promoter region. See FIG. 8, Step 1. This fourthamplicon is used a template to transcribe multiple copies of an RNAamplicon complementary to the fourth amplicon, not including thepromoter portion, using an RNA polymerase that recognizes thedouble-stranded promoter and initiates transcription therefrom. See FIG.8, Steps 2 and 3. The resulting RNA amplicons comprise segments cT andcD1_(p), complementary to the T1 and D1_(p) priming sequences, and maythus be used as templates for further amplification using the third andfourth amplification oligomers comprising priming segments T1_(p) andD1_(p), respectively. Amplicons produced by extension from T1_(p) orD1_(p) on the RNA amplicon template may then serve as templates forfurther amplification upon hybridization of the second amplificationoligomer via segment T2, formation of a double-stranded promoter, andRNA transcription as summarized above.

In other embodiments employing TMA, a promoter provider is used is usedin the amplification reaction. FIG. 9 illustrates such a variation, inwhich the target nucleic acid is contacted with a terminationoligonucleotide (TERM) in addition to a first amplificationoligonucleotide comprising priming segment T1 and displacer tag D1. SeeFIG. 9, Step 1. The terminating oligonucleotide hybridizes to a targetsequence that is adjacent to the 5′-end of the target region, such thatextension of the first amplification oligomer (shown in FIG. 9 ascomprising priming segment T1, displacer tag D1, and spacer S1) isterminated at the 3′-end of the terminating oligonucleotide, therebyproviding a defined 3′-end for the first amplicon (API) that correspondsto the 5′-end of the target region. See id., Steps 1 and 2. The firstamplicon is then separated from the target sequence (e.g., using anenzyme that selectively degrades that target sequence, such as, forexample, RNAse H for an RNA target nucleic acid). See id, Step 3. Thefirst amplicon is then contacted with a second amplificationoligonucleotide comprising target-binding segment T2, substantiallycomplementary to the defined 3′-end of the first amplicon, and apromoter sequence P situated 5′ to T2. See id., Step 4. The secondamplification oligomer is modified to prevent initiation of DNAsynthesis, preferably by situating a blocking moiety (X) at the 3′-endof the oligonucleotide. See id. The T2 segment of the secondamplification oligonucleotide hybridizes to the 3′-end of the firstamplicon, and the 3′-end of the first amplicon is extended to addsegment cP having a sequence complementary to the promoter sequence P,resulting in the formation of a double-stranded promoter sequence. Seeid., Steps 4 and 5. The first amplicon is used a template to transcribemultiple copies of a second amplicon AP2 complementary to the firstamplicon, not including the promoter portion, using an RNA polymerasethat recognizes the double-stranded promoter and initiates transcriptiontherefrom. See id., Steps 6 and 7. As in the previous example depictedin FIG. 8, the resulting RNA amplicons comprise segments cT and cD1_(p),complementary to the T1 and D1_(p) priming sequences, and may thus beused as templates for further amplification using third and fourthamplification oligomers comprising priming segments T1_(p) and D1_(p),respectively. Amplicons produced by extension from T1_(p) or D1_(p) onthe AP2 template may then serve as templates for further amplificationupon hybridization of the second amplification oligomer via segment T2,formation of a double-stranded promoter, and RNA transcription.

In more particular variations of embodiments employing TMA, a promoterprimer or promoter primer further includes one or more displacer tagssituated 5′ to a target-binding segment. In such embodiments, the one ormore displacer tag(s) of a promoter primer or promoter primer arepreferably situated 3′ to the promoter sequence. In this manner,extension of the 3′-end of the template amplicon, upon hybridization oftarget-binding segment T2, produces a template comprising segmentscomplementary to the displacer tag(s) and incorporation of the displacertag(s) into the resulting RNA amplicon. Subsequent amplification of theRNA amplicon may then further include the use of displaceroligonucleotides to initiate displacement reactions in accordance withthe present invention.

In some embodiments of the method, amplification of target regionutilizes a target-specific priming site situated downstream from (5′ to)a displacer priming site. For example, in particular variationsutilizing a T1-D1 first amplification oligomer, a third amplificationoligomer comprises a priming segment T1_(p) having a nucleotide sequencesubstantially corresponding to T1 (and hence configured to hybridize tocT1, see, e.g., FIGS. 1A and 1B.) In alternative variations, atarget-specific priming sequence substantially corresponds to thecomplement of a target sequence near or overlapping with the initialtarget sequence recognized by the target-binding segment of adisplacer-tagged amplification oligomer. For example, in alternativevariations of the amplification reaction depicted in FIGS. 1A and 1B,the third amplification oligomer T1_(p) can be configured to hybridizeto a target-specific sequence cT1′ that is within the second ampliconAP2 and is different from cT1 (i.e., a sequence different from thatcorresponding to the initial target-specific priming site), wherein cT1′is situated within the target region 5′ to cD1 and is near oroverlapping with cT1. Similarly, e.g., in alternative variations of theamplification reaction depicted in FIG. 7, utilizing a displacer-taggedreverse amplification oligomer comprising a target-binding segment T2,an amplification oligomer T2_(p) can be configured to hybridize to atarget-specific sequene cT2′ that is within the fourth amplicon AP4 andis different from cT2, wherein cT2′ is near or overlapping with cT2 andis situated 5′ to cD4. More preferably, the 3′-terminal base of thedisplacer oligonucleotide is spaced from 5 to 35 bases from the5′-terminal base of the forward priming oligonucleotide. A targetsequence is “near” a reference target sequence (e.g., near cT1 or cT2)if the 3′-end of the target sequence is within 50 bases, preferablywithin 40 bases, more preferably within 30 bases, and most preferablywithin 20, within 10, or within 5 bases of the 5′-end of the referencetarget sequence.

As previously discussed, certain variations of the method incorporatethe use of universal priming sites and primers following initialamplification of a target nucleic acid. In such embodiments, anamplification oligomer comprising a target-specific hybridizing sequence(e.g., target-binding segment T1 of a first amplificationoligonucleotide, or target-binding segment T2 of a second amplificationoligonucleotide) includes a heterologous universal tag segment situated5′ to the target-specific segment T1. In this manner, subsequent roundsof amplification can employ the use of an amplification oligonucleotidecomprising a universal priming segment (e.g., “U1_(p)”) corresponding tothe universal tag, in place of an oligonucleotide comprising atarget-binding priming segment (e.g., “T1_(p)”) specific for the targetregion of the target nucleic acid. In certain embodiments, both aforward and a reverse amplification oligomer (e.g., first and secondamplification oligonucleotides as discussed herein) include a universaltag situated 5′ to a target-binding segment. In particular embodimentsemploying a promoter primer or promoter primer for TMA, a universal tagis included 5′ to the target-binding segment and 3′ to the promotersequence. Any variation of the methods discussed herein can be adaptedfor use with universal priming sites and primers following initialamplification of a target nucleic acid and such embodiments are withinthe scope of the present invention.

In each of the embodiments described herein, a wide variety ofidentities and functionalities can be designed into the displacer tagsequences. For example, in some embodiments, where two or more displacersequences are used, at least two of these sequence can be different fromeach other. In other embodiments, at least two of the multiple displacertags can be the same as each other. Similarly, in some variations wherea universal priming sequence is used, one or more displacer tagsequence(s) is the same as the universal sequence; in other variations,one or more of the displacer tag sequence(s) are different from theuniversal sequence. Thus, in some embodiments, wherever two or moreheterologous sequences (universal and displacer) are employed, there canbe at least two unique sequences, or more depending on the number ofdisplacer sequences added. In a more specific variation employingmultiple displacer tags with a universal tag, each of the multipledisplacer tags sequences are the same but different from the universalsequence.

In other more particular variations, sequences are designed to havedifferent affinities for their complements. For example, in certainembodiments the affinity of a first displacer tag D1 for its complementis lower than that of a target-specific site T1 (or lower than that of auniversal site U1 if a universal tag is used). Similarly, where multipledisplacers are used, each successive displacer may be designed to have alower affinity for its complement that the one situate 3′ to it (e.g.,D2 can be designed to have a lower affinity for its complement than D1;D3 can be designed to have a lower affinity for its complement than D2;etc.). In this way, the displacing potential of the oligonucleotideconstructs can be increased by increasing, for example, the potentialthat a target-specific or universal priming site is available forbinding before binding and extension of a D1 displacer oligomer (e.g.,allowing a D1 displacer oligomer to bind to its priming site “after”binding of a target-specific or universal primer having a higheraffinity), thereby maximizing the potential for binding and extensionfrom both priming oligonucleotides together with a displacementreaction. The skilled artisan will appreciate that affinities of variousdisplacer and universal sequences may be varied as desired based uponsuch known factors as, e.g., GC content and length of the hybridizingsequence.

In addition to improving the kinetics of an amplification reaction viadisplacement of extension products from their template strands,tag-mediated displacement reactions can also increase amplificationcapacity. This advantage of the present invention is illustrated in FIG.10, which shows a particular embodiment of a tag-mediated displacementstrategy utilizing a universal priming site cU1 and a displacer primingsite cD1 incorporated into an amplicon, together with three primingoligonucleotides—universal priming oligomer U1_(p), substantiallycomplementary to cU1; displacer priming oligonucleotide D1_(p),substantially complementary to cD1; and a priming oligonucleotidecomprising priming segment D1_(p), optional spacer S2, and heterologousdisplacer tag D2 (the “D1_(p)-D2 oligomer”). See FIG. 10, Step 1.Extension reactions initiated from these three amplification oligomersyields up to three potential species as shown in Step 2. Each of thesepotential species can be further amplified in the next round to producethe three complementary amplicons as depicted in Step 3. In thisexample, in the next round of amplification, four amplificationoligomers are depicted for use in further amplification: the U1_(p)oligomer, the D1_(p) oligomer, the D1_(p)-D2 oligomer, and a fourtholigomer which is a displacer priming oligonucleotide D2_(p). In such ascenario, in the next amplification round, the first complementaryamplicon can bind U1_(p) to produce one amplicon; the secondcomplementary amplicon can bind U1_(p) together with D1_(p) or D1_(p)-D2to produce up to two amplicons; and the third complementary amplicon canbind U1_(p) together with D1_(p) and D2_(p), or together with D1_(p)-D2,to yield up to three amplicons, thereby yielding up to six amplicons inthe next round of the amplification reaction. See FIG. 10, Steps 3 and4.

The methods of the present invention are useful in assays for detectingand/or quantitating specific target nucleic acids in clinical, water,environmental, industrial, beverage, food, seed stocks, and othersamples or to produce large numbers of nucleic acid amplificationproducts from a specific target sequence for a variety of uses. Forexample, the present invention is useful to screen clinical samples(e.g., blood, urine, feces, saliva, semen, or spinal fluid), food,water, laboratory and/or industrial samples for the presence of specificnucleic acids, specific organisms (e.g., using species-specificoligonucleotides) and/or specific classes of organisms (e.g., usingclass-specific oligonucleotides) in applications such as in sterilitytesting. The present invention can be used to detect the presence of,for example, viruses, bacteria, fungi, or parasites.

Samples suspected of containing a target nucleic acid are prepared forsubsequent amplification as described herein using methods generallyknown in the art. Typically, a target nucleic acid is separated orpurified from one or more other components of a sample. Suchpurification may include may include methods of separating and/orconcentrating organisms contained in a sample from other samplecomponents. In particular embodiments, purifying the target nucleic acidincludes capturing the target nucleic acid to specifically ornon-specifically separate the target nucleic acid from other samplecomponents. Non-specific target capture methods may involve selectiveprecipitation of nucleic acids from a substantially aqueous mixture,adherence of nucleic acids to a support that is washed to remove othersample components, or other means of physically separating nucleic acidsfrom a mixture that contains target nucleic acid and other samplecomponents.

In some embodiments, a target nucleic acid is selectively separated fromother sample components by specifically hybridizing the target nucleicacid to a capture probe oligomer. The capture probe comprises atarget-binding segment configured to specifically hybridize to a targetsequence so as to form a target-nucleic-acid:capture-probe(“target:capture-probe”) complex that is separated from samplecomponents. In a preferred variation, the specific target capturefurther comprises binding the target:capture-probe complex to animmobilized probe to form a target:capture-probe:immobilized-probecomplex that is separated from the sample and, optionally, washed toremove non-target sample components (see, e.g., U.S. Pat. Nos.6,110,678; 6,280,952; and 6,534,273; each incorporated by referenceherein). In such variations, the capture probe oligomer furthercomprises a sequence or moiety that binds attaches the capture probe,with its bound target-binding segment, to an immobilized probe attachedto a solid support, thereby permitting the hybridized target nucleicacid to be separated from other sample components. In more specificembodiments, the capture probe oligomer includes a tail portion (e.g., a3′ tail) that is not complementary to the target nucleic acid but thatspecifically hybridizes to a nuclei acid sequence on the immobilizedprobe, thereby serving as the moiety allowing the target nucleic acid tobe separated from other sample components, such as previously describedin, e.g., U.S. Pat. No. 6,110,678, incorporated herein by reference. Anysequence may be used in a tail region, which is generally about 5 to 50nt long, and preferred embodiments include a substantially homopolymerictail of about 10 to 40 nt (e.g., A₁₀ to A₄₀), more preferably about 14to 33 nt (e.g., A₁₄ to A₃₀ or T₃A₁₄ to T₃A₃₀), that bind to acomplementary immobilized sequence (e.g., poly-T) attached to a solidsupport, e.g., a matrix or particle.

Target capture typically occurs in a solution phase mixture thatcontains one or more capture probes that hybridize specifically to thetarget nucleic acid under hybridizing conditions, usually at atemperature higher than the T_(m) of thetail-sequence:immobilized-probe-sequence duplex. For embodimentscomprising a capture probe tail, the target:capture-probe complex iscaptured by adjusting the hybridization conditions so that the captureprobe tail hybridizes to the immobilized probe, and the entire complexon the solid support is then separated from other sample components. Thesupport with the attached target:capture-probe:immobilized-probe may bewashed one or more times to further remove other sample components.Certain embodiments use a particulate solid support, such asparamagnetic beads, so that particles with the attachedtarget:capture-probe:immobilized-probe complex may be suspended in awashing solution and retrieved from the washing solution, preferably byusing magnetic attraction. To limit the number of handling steps, thenucleic acid target region may be amplified by simply mixing the targetnucleic acid in the complex on the support with amplification oligomersand proceeding with amplification steps.

For amplification of a target nucleic acid, a variety of nucleic acidamplification methods are known and may be readily adapted for use toincorporate a tag-mediated displacement strategy in accordance with thepresent invention (see above). Generally, certain amplification steps asdescribed herein are “extension reactions” in which the 3′-end of apriming oligonucleotide is extended by the addition of nucleotidescomplementary to a nucleic acid template to which the primingoligonucleotide is hybridized, thereby synthesizing a complementary copyof the template. Conditions for extension reactions are well-known andgenerally utilize a polymerization agent (e.g., DNA polymerase) tosynthesize the complementary DNA copy. A DNA polymerase may becharacterized as “DNA-dependent” or “RNA-dependent,” depending onwhether the polymerase utilizes a DNA template or RNA template,respectively. Examples of DNA-dependent DNA polymerases are DNApolymerase I from E. coli, bacteriophage T7 DNA polymerase, or DNApolymerases from bacteriophages T4, Phi-29, M2, or T5. DNA-dependent DNApolymerases may be the naturally occurring enzymes isolated frombacteria or bacteriophages or expressed recombinantly, or may bemodified or “evolved” forms which have been engineered to possesscertain desirable characteristics, e.g., thermostability, or the abilityto recognize or synthesize a DNA strand from various modified templates.It is known that under suitable conditions a DNA-dependent DNApolymerase may synthesize a complementary DNA copy from an RNA template.Alternatively, in some variations, an RNA-dependent DNA polymerase, or“reverse transcriptase” (“RT”), is utilized in a primer extensionreaction. A reverse transcriptase synthesizes a complementary DNA copyfrom an RNA template. All known reverse transcriptases also have theability to make a complementary DNA copy from a DNA template, and arethus both RNA- and DNA-dependent. RTs may also have an RNAse H activity.

Other amplification steps as described herein do not necessarily requirean extension reaction. For example, certain amplification reactions maybe catalyzed using a DNA-dependent RNA polymerase or “transcriptase,”which synthesizes multiple RNA copies from a double-stranded orpartially double-stranded DNA molecule having a promoter sequence thatis usually double-stranded. The RNA molecules (“transcripts”) aresynthesized in the 5′-to-3′ direction beginning at a specific positionjust downstream of the promoter. Examples of transcriptases are theDNA-dependent RNA polymerase from E. coli and bacteriophages T7, T3, andSP6. As previously discussed, in certain embodiments, a promoter regionis introduced into an amplification reaction by the use of anamplification oligomer further comprising a promoter sequence situated5′ to a target-binding segment.

Certain embodiments of the present invention relate to amplification ofa target nucleic acid utilizing transcription-mediated amplification(TMA). Some such embodiments relate more specifically to amplificationof a target nucleic acid comprising an RNA target region. In certaincases, the target nucleic acid has indeterminate 3′- and 5′-endsrelative to the desired RNA target region. The target nucleic acid istreated with a priming oligonucleotide which has a base regionsufficiently complementary to a 3′-end of the RNA target region tohybridize therewith and, as discussed above, further comprises at leastone heterologous displacer tag and optionally a universal tag in thefirst primer extension reaction. Priming oligonucleotides are designedto hybridize to a suitable region of any desired target sequence,according to primer design methods well-known to those of ordinary skillin the art. While the presence of the displacer tag sequence in apriming oligonucleotide may alter the binding characteristics of atarget hybridizing region to a target nucleic acid sequence, the artisanskilled in the molecular arts can readily design primingoligonucleotides which contain both a target-binding segment and tagsegments that can be used in accordance with the methods describedherein. Additionally, the 5′-end of a priming oligonucleotide(preferably not a tagged priming oligonucleotide) may include one ormodifications which improve the binding properties (e.g., hybridizationor base stacking) of the priming oligonucleotide to a DNA extensionproduct or to an RNA amplification product, provided the modificationsdo not substantially interfere with the priming function of the primingoligonucleotide or cleavage of an RNA amplification product to which thepriming oligonucleotide is hybridized. The 3′-end of the primingoligonucleotide is extended by an appropriate DNA polymerase, e.g., anRNA-dependent DNA polymerase (reverse transcriptase) in an extensionreaction using the RNA target region or amplification product as atemplate to give a DNA primer extension product which is complementaryto the RNA template or amplification product.

DNA primer extension products may be separated (at least partially) froman RNA template using an enzyme which degrades the RNA template oramplification product. Suitable enzymes, i.e., “selective RNAses,” arethose which act on the RNA strand of an RNA:DNA complex, and includeenzymes which comprise an RNAse H activity. Some reverse transcriptasesinclude an RNAse H activity, including those derived from Moloney murineleukemia virus and avian myeloblastosis virus. According to preferredamplification embodiments, the selective RNAse may be provided as anRNAse H activity of a reverse transcriptase, or may be provided as aseparate enzyme, e.g., as an E. coli RNAse H or a T. thermophilus RNAseH. Other enzymes which selectively degrade RNA present in an RNA:DNAduplex may also be used.

Following initial amplification of an RNA target region so as toincorporate one or more displacer tags into an amplification product,subsequent strand separation may be achieved using displaceroligonucleotides corresponding to the displacer tag sequences asdescribed herein. For example, referring FIG. 9, which depicts the useof a promoter provider in transcription-mediated amplification of atarget nucleic acid (which may be, e.g., an RNA target nucleic acid),multiple RNA amplicons (AP2) are produced comprising the complement ofthe D1 displacer tag (cD1) situated 5′ to the complement of the T1priming segment (cT1). See FIG. 9, Step 7. Subsequent amplification ofan AP2 amplicon may utilize both T1_(p) and D1_(p) primingoligonucleotides substantially complementary to cT1 and cD1,respectively, such as previously illustrated, e.g., in FIGS. 1A and 1B.Briefly, referring to the further amplification of AP2 depicted in FIGS.1A and 1B, in the presence of a DNA polymerase, the 3′-end of the T1_(p)priming oligonucleotide is extended in a template-dependent manner toform a one extension product (a “third amplicon” or “AP3”), while the3′-end of the D1_(p) displacer oligonucleotide is also extended to formanother extension product (“AP4”) that displaces the AP3 extensionproduct from the target nucleic acid (see Steps 7 to 9). In this manner,an extension product produced on an RNA amplicon template is madeavailable for hybridization to an amplification oligomer for furtheramplification (e.g., hybridization to a promoter primer or promoterprovider oligonucleotide) without the need for RNAse-mediateddegradation of the RNA template.

When the initial target nucleic acid is DNA, then a first forwardamplification oligomer can be a DNA primer comprising a 3′target-binding priming segment and a 5′ first heterologous displacertag. The first forward amplification oligomer can be used to make afirst DNA primer extension product. When the amplification reaction is aPCR amplification reaction, then the first DNA primer extension productcan be separated from the target nucleic acid by a number of methods,including using the temperature cycling parameters of a PCR reaction.Alternatively, and also for use with isothermal amplification reactions,the first DNA primer extension product can be separated from the fromthe target nucleic acid using a displacer primer comprising atarget-specific priming segment that hybridizes to the DNA targetnucleic acid at a position upstream from the forward primingoligonucleotide binding site (a first amplification oligomer such asdescribed herein). In this manner, the first amplicon produced byextension of the first amplification oligomer can be displaced from thetemplate strand by extension of the target-specific displacer primer,thereby making it available for hybridization to an amplificationoligonucleotide (e.g., a promoter primer or promoter provider, or otheramplification oligomer as described herein) for further amplification toproduce a second amplicon. In a further alternative approach, conditionscan be established whereby an oligonucleotide gains access to the firstDNA primer extension product through strand invasion facilitated by, forexample, DNA breathing (e.g., AT rich regions), low salt conditions,and/or the use of DMSO and/or osmolytes, such as betaine. A particularlysuitable amplification oligomer for this embodiment is a promoterprovider oligonucleotide such as that described herein, which ismodified to prevent the promoter oligonucleotide from functioning as apriming oligonucleotide for a DNA polymerase (e.g., the promoteroligonucleotide includes a blocking moiety at its 3′-terminus).Irrespective of the method used to separate the first amplicon from aDNA template, once the first amplicon is made available to produce asecond amplicon and with the use of heterologous displacer tags toincorporate heterologous displacer priming sites in amplicons asdescribed herein, tag-mediated displacement can be utilized insubsequent amplification rounds to separate nucleic acid strands.

In certain embodiments, the methods of the present invention furthercomprise treating a target nucleic acid as described above to limit thelength of a primer extension product to a certain desired length. Suchlength limitation is typically carried out through use of a “bindingmolecule” which hybridizes to or otherwise binds to the target nucleicacid adjacent to or near the 5′-end of the desired target sequence. Incertain embodiments, a binding molecule comprises a base region. Thebase region may be DNA, RNA, a DNA:RNA chimeric molecule, or an analogthereof Binding molecules comprising a base region may be modified inone or more ways, as described elsewhere herein. Suitable bindingmolecules include, but are not limited to, a binding molecule comprisinga terminating oligonucleotide or a terminating protein that binds RNAand/or DNA and prevents primer extension past its binding region, or abinding molecule comprising a modifying molecule, for example, amodifying oligonucleotide such as a “digestion” oligonucleotide thatdirects hydrolysis of that portion of the RNA and/or DNA targethybridized to the digestion oligonucleotide, or a sequence-specificnuclease that cuts the RNA and/or DNA target.

Illustrative terminating oligonucleotides of the present invention havea 5′-base region sufficiently complementary to the target nucleic acidat a region adjacent to, near to, or overlapping with the 5′-end of thetarget sequence, to hybridize therewith. In certain embodiments, aterminating oligonucleotide is synthesized to include one or moremodified nucleotides. For example, certain terminating oligonucleotidesof the present invention comprise one or more 2′-O-ME ribonucleotides,or are synthesized entirely of 2′-O-ME ribonucleotides. See, e.g.,Majlessi et al., Nucleic Acids Res., 26, 2224-2229, 1998. A terminatingoligonucleotide of the present invention typically also comprises ablocking moiety at its 3′-end to prevent the terminating oligonucleotidefrom functioning as a primer for a DNA polymerase. In some embodiments,the 5′-end of a terminating oligonucleotide of the present inventionoverlaps with and is complementary to at least about 2 nucleotides ofthe 5′-end of the target region. Typically, the 5′-end of a terminatingoligonucleotide of the present invention overlaps with and iscomplementary to at least 3, 4, 5, 6, 7, or 8 nucleotides of the 5′-endof the target sequence, but no more than about 10 nucleotides of the5′-end of the target region. (As used herein, the term “end” refers to a5′- or 3′-region of an oligonucleotide, nucleic acid or nucleic acidregion which includes, respectively, the 5′- or 3′-terminal base of theoligonucleotide, nucleic acid or nucleic acid region.)

In particular embodiment employing transcription-mediated amplification,a single-stranded DNA primer extension product, or “first” DNA primerextension product, which has either a defined 3′-end or an indeterminate3′-end, is treated with a promoter oligonucleotide (a promoter primer orpromoter provider) that comprises a target-binding segment substantiallycomplementary to a 3′-region of the DNA primer extension product tohybridize therewith, and a second segment comprising a promoter for anRNA polymerase, e.g., T7 polymerase, which is situated 5′ to the firstsegment (e.g., immediately 5′ to or spaced from the first region). Insome variations, the promoter oligonucleotide is a promoter providermodified to prevent the promoter oligonucleotide from functioning as aprimer for a DNA polymerase (e.g., the promoter oligonucleotide includesa blocking moiety attached at its 3′-terminus). In particularvariations, a promoter oligonucleotide further includes one or moreheterologous displacer tags situated 5′ to the target-binding segmentand, most typically, 3′ to the promoter region. In other embodiments, apromoter oligonucleotide includes a universal tag segment 5′ to thetarget-binding segment and 3′ to the promoter and any displacer tag(s).Upon identifying a desired target-binding segment and any desireddisplacer or universal tags, suitable promoter oligonucleotides can beconstructed by one of ordinary skill in the art using only routineprocedures. Those of ordinary skill in the art will readily understandthat a promoter region has certain nucleotides which are required forrecognition by a given RNA polymerase. In addition, certain nucleotidevariations in a promoter sequence might improve the functioning of thepromoter with a given enzyme, including the use of an intervening spacersegment between the promoter sequence and the target-binding segment(also referred to as an “insertion sequence”). Insertion sequences maybe positioned between the target-binding and promoter segments ofpromoter oligonucleotides and function to increase amplification rates.(A displacer or universal tag segment of a tagged promoteroligonucleotide may provide this beneficial effect.)

Assaying promoter oligonucleotides with variations in the promotersequences is easily carried out by the skilled artisan using routinemethods. Furthermore, if it is desired to utilize a different RNApolymerase, the promoter sequence in the promoter oligonucleotide iseasily substituted by a different promoter. Substituting differentpromoter sequences is well within the understanding and capabilities ofthose of ordinary skill in the art. For real-time TMA, promoteroligonucleotides provided to the amplification reaction mixture aremodified to prevent efficient initiation of DNA synthesis from their3′-termini, and preferably comprise a blocking moiety attached at their3′-termini. Furthermore, terminating oligonucleotides and cappingoligonucleotides, and even probes used in certain embodiments of thepresent invention also optionally comprise a blocking moiety attached attheir 3′-termini.

Where a terminating oligonucleotide is used in a TMA reaction, thefirst, target-binding segment of a promoter oligonucleotide is designedto hybridize with a desired 3′-end of the first DNA primer extensionproduct with substantial, but not necessarily exact, precision.Subsequently, the second segment of the promoter oligonucleotide may actas a template, allowing the first DNA primer extension product to befurther extended to add a base region complementary to the secondsegment of the promoter oligonucleotide, i.e., the segment comprisingthe promoter sequence, rendering the promoter double-stranded.Alternatively, where a terminating oligonucleotide or other bindingmolecule is not used in a TMA reaction, a promoter primer may be used asthe promoter oligonucleotide, thereby allowing the incorporation of apromoter sequence into a second extension product initiated from andthus comprising the promoter primer. In this case, priming of a thirdextension product using the second extension product as a templateproduces a double-stranded DNA that includes the double-strandedpromoter. An RNA polymerase which recognizes the promoter then binds tothe promoter sequence, and initiates transcription of multiple RNAcopies complementary to the DNA primer extension product, which copiesare substantially identical to the target region. By “substantiallyidentical” it is meant that the multiple RNA copies may have additionalnucleotides either 5′ or 3′ relative to the target sequence, or may havefewer nucleotides either 5′ or 3′ relative to the target sequence,depending on, e.g., the boundaries of the target region, thetranscription initiation point, or whether the priming oligonucleotidecomprises additional nucleotides 5′ of the primer region. Where a targetregion is DNA, the sequence of the RNA copies is described herein asbeing “substantially identical” to the target region. It is to beunderstood, however, that an RNA sequence which has uridine residues inplace of the thymidine residues of the DNA target region still has a“substantially identical” sequence. The RNA transcripts so produced mayautomatically recycle in the above system without further manipulation.Thus, this reaction is autocatalytic.

Promoters or promoter sequences suitable for incorporation in promoteroligonucleotides used in the methods of the present invention arenucleic acid sequences (either naturally occurring, producedsynthetically or a product of a restriction digest) that arespecifically recognized by an RNA polymerase that recognizes and bindsto that sequence and initiates the process of transcription, whereby RNAtranscripts are produced. Typical, known and useful promoters includethose which are recognized by certain bacteriophage polymerases, such asthose from bacteriophage T3, T7, and SP6, and a promoter from E. coli.The sequence may optionally include nucleotide bases extending beyondthe actual recognition site for the RNA polymerase which may impartadded stability or susceptibility to degradation processes or increasedtranscription efficiency. Promoter sequences for which there is a knownand available polymerase that is capable of recognizing the initiationsequence are particularly suitable to be employed.

Suitable DNA polymerases for use in accordance with the methods of theinvention, particularly for use with embodiments employing TMA, includereverse transcriptases. Particularly suitable DNA polymerases includeAMV reverse transcriptase and MMLV reverse transcriptase. Some of thereverse transcriptases suitable for use in the methods of the presentinvention, such as AMV and MMLV reverse transcriptases, have an RNAse Hactivity. Indeed, according to certain embodiments of the presentinvention, the only selective RNAse activity in the amplificationreaction is provided by the reverse transcriptase—no additionalselective RNAse is added. However, in some situations it may also beuseful to add an exogenous selective RNAse, such as E. coli RNAse H.Although the addition of an exogenous selective RNAse is not required,under certain conditions, the RNAse H activity present in, e.g., AMVreverse transcriptase may be inhibited or inactivated by othercomponents present in the reaction mixture. In such situations, additionof an exogenous selective RNAse may be desirable. For example, whererelatively large amounts of heterologous DNA are present in the reactionmixture, the native RNAse H activity of the AMV reverse transcriptasemay be somewhat inhibited and thus the number of copies of the targetsequence produced accordingly reduced. In situations where the targetnucleic acid comprises only a small portion of the nucleic acid present(e.g., where the sample contains significant amounts of heterologous DNAand/or RNA), it is particularly useful to add an exogenous selectiveRNAse. See, e.g., Kacian et al, U.S. Pat. No. 5,399,491, incorporated byreference herein.

RNA amplification products produced by TMA methods may serve astemplates to produce additional amplification products related to thetarget sequence through mechanisms described herein. The system isautocatalytic and amplification by the methods of the present inventionoccurs without the need for repeatedly modifying or changing reactionconditions such as temperature, pH, ionic strength and the like. Thesemethods do not require an expensive thermal cycling apparatus, nor dothey require several additions of enzymes or other reagents during thecourse of an amplification reaction.

The amplification product can be detected by any conventional means. Forexample, amplification product can be detected by hybridization with adetectably labeled probe and measurement of the resulting hybrids.Design criteria in selecting probes for detecting particular targetsequences are well-known in the art and are described in, for example,Hogan et al., U.S. Pat. No. 6,150,517, incorporated by reference herein.Generally, probes should be designed to maximize homology for the targetsequence(s) and minimize homology for possible non-target sequences. Tominimize stability with non-target sequences, guanine and cytosine richregions should be avoided, the probe should span as many destabilizingmismatches as possible, and the length of perfect complementarity to anon-target sequence should be minimized. Contrariwise, stability of theprobe with the target sequence(s) should be maximized, adenine andthymine rich regions should be avoided, probe:target hybrids arepreferably terminated with guanine and cytosine base pairs, extensiveself-complementarity is generally to be avoided, and the meltingtemperature of probe:target hybrids should be about 2-10° C. higher thanthe assay temperature.

In a particular embodiment, the amplification product is assayed by theHybridization Protection Assay (“HPA”), which involves hybridizing achemiluminescent oligonucleotide probe (e.g., an acridiniumester-labeled (“AE”) probe) to its target sequence, selectivelyhydrolyzing the chemiluminescent label present on unhybridized probe,and measuring the chemiluminescence produced from the remaining probe ina luminometer. See, e.g., Arnold et al., U.S. Pat. No. 5,283,174 andNorman C. Nelson et al., Nonisotopic Probing, Blotting, and Sequencing,Ch. 17 (Larry J. Kricka ed., 2d ed. 1995), each incorporated byreference herein.

In further embodiments, the present invention provides quantitativeevaluation of the amplification process in real-time. Evaluation of anamplification process in “real-time” involves determining the amount ofamplicon in the reaction mixture either continuously or periodicallyduring the amplification reaction, and the determined values are used tocalculate the amount of target sequence initially present in the sample.There are a variety of known methods for determining the amount ofinitial target sequence present in a sample based on real-timeamplification. These include those disclosed by Wittwer et al., U.S.Pat. No. 6,303,305, and Yokoyama et al., U.S. Pat. No. 6,541,205, eachincorporated by reference herein. Another method for determining thequantity of target sequence initially present in a sample, but which isnot based on a real-time amplification, is disclosed by Ryder et al.,U.S. Pat. No. 5,710,029, incorporated by reference herein.

Amplification products may be detected in real-time through the use ofvarious self-hybridizing detection probes, most of which have astem-loop structure. Such self-hybridizing probes are labeled so thatthey emit differently detectable signals, depending on whether thedetection probes are in a self-hybridized state or an altered statethrough hybridization to a target sequence. By way of example,“molecular torches” are a type of self-hybridizing detection probe thatincludes distinct regions of self-complementarity (referred to as “thetarget binding domain” and “the target closing domain”) that areconnected by a joining region (e.g., non-nucleotide linker) andhybridize to each other under predetermined hybridization assayconditions. In a preferred embodiment, molecular torches containsingle-stranded base regions in the target-binding domain that are from1 to about 20 bases in length and are accessible for hybridization to atarget sequence present in an amplification product under stranddisplacement conditions. Under strand displacement conditions,hybridization of the two complementary regions (which may be fully orpartially complementary) of the molecular torch is favored, except inthe presence of the target sequence, which will bind to thesingle-stranded region present in the target-binding domain and displaceall or a portion of the target closing domain. The target binding domainand the target closing domain of a molecular torch include a detectablelabel or a pair of interacting labels (e g., luminescent/quencher)positioned so that a different signal is produced when the moleculartorch is self-hybridized than when the molecular torch is hybridized tothe target sequence, thereby permitting detection of probe:targetduplexes in a test sample in the presence of unhybridized moleculartorches. Molecular torches and a variety of types of interacting labelpairs are disclosed by Becker et al., U.S. Pat. No. 6,534,274,incorporated by reference herein.

Another example of a detection probe having self-complementarity is a“molecular beacon.” Molecular beacons include nucleic acid moleculeshaving a target complement sequence, an affinity pair (or nucleic acidarms) holding the probe in a closed conformation in the absence of atarget sequence present in an amplification product, and a label pairthat interacts when the probe is in a closed conformation. Hybridizationof the target sequence and the target complement sequence separates themembers of the affinity pair, thereby shifting the probe to an openconformation. The shift to the open conformation is detectable due toreduced interaction of the label pair, which may be, for example, afluorophore and a quencher (e.g., DABCYL and EDANS). Molecular beaconsare disclosed by Tyagi et al., U.S. Pat. No. 5,925,517, and Tyagi etal., U.S. Pat. No. 6,150,097, each incorporated by reference herein.

Other self-hybridizing probes for use in the present invention arewell-known to those of ordinary skill in the art. By way of example,probe binding pairs having interacting labels, such as those disclosedby Morrison, U.S. Pat. No. 5,928,862, and Gelfand et al., U.S. Pat. No.5,804,375 for PCR reactions (each incorporated by reference herein),might be adapted for use in the present invention. Additional detectionsystems include “molecular switches,” as disclosed by Arnold et al.,U.S. Pat. Appln. Pub. No. US 2005-0042638 A1, incorporated by referenceherein. And other probes, such as those comprising intercalating dyesand/or fluorochromes, might be useful for detection of amplificationproducts in the present invention. See, e.g., Ishiguro et al., U.S. Pat.No. 5,814,447, incorporated by reference herein.

In those methods of the present invention where the initial targetsequence and the RNA transcription product share the same sense, it maybe desirable to initiate amplification before adding probe for real-timedetection. Adding probe prior to initiating an amplification reactionmay slow the rate of amplification since probe which binds to theinitial target sequence has to be displaced or otherwise remove duringthe primer extension step to complete a primer extension product havingthe complement of the target sequence. The initiation of amplificationis judged by the addition of amplification enzymes (e.g., a reversetranscriptase and an RNA polymerase).

Also provided by the subject invention is a reaction mixture foramplification of a target nucleic acid. A reaction mixture in accordancewith the present invention at least comprises a combination ofamplification oligomers as described herein for amplification of anucleic acid target region. In certain embodiments, a reaction mixturealso includes a capture probe for purifying the target nucleic acidand/or a detection probe for determining the presence or absence of anamplification product. The reaction mixture may further include a numberof optional components such as, for example, arrays of capture probenucleic acids. For an amplification reaction mixture, the reactionmixture will typically include other reagents suitable for performing invitro amplification such as, e.g., buffers, salt solutions, appropriatenucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTPand UTP), and/or enzymes (e.g., reverse transcriptase, and/or RNApolymerase), and will typically include test sample components, in whicha target nucleic acid may or may not be present. In addition, for areaction mixture that includes a detection probe together with anamplification oligomer combination, selection of amplification oligomersand detection probe oligomers for a reaction mixture are linked by acommon target region (i.e., the reaction mixture will include a probethat binds to a sequence amplifiable by an amplification oligomercombination of the reaction mixture).

Also provided by the subject invention are kits for practicing themethods as described herein. A kit in accordance with the presentinvention at least comprises a combination of amplification oligomers asdescribed herein for amplification of a nucleic acid target region. Incertain embodiments, a reaction mixture also includes a capture probefor purifying the target nucleic acid and/or a detection probe fordetermining the presence or absence of an amplification product. Thekits may further include a number of optional components such as, forexample, arrays of capture probe nucleic acids. Other reagents that maybe present in the kits include reagents suitable for performing in vitroamplification such as, e.g., buffers, salt solutions, appropriatenucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTPand UTP), and/or enzymes (e.g., reverse transcriptase, and/or RNApolymerase). Oligomers as described herein may be packaged in a varietyof different embodiments, and those skilled in the art will appreciatethat the invention embraces many different kit configurations. Forexample, a kit may include amplification oligomers for only one targetregion, or it may include amplification oligomers for multiple targetregions. In addition, for a kit that includes a detection probe togetherwith an amplification oligomer combination, selection of amplificationoligomers and detection probe oligomers for a kit are linked by a commontarget region (i.e., the kit will include a probe that binds to asequence amplifiable by an amplification oligomer combination of thekit). In certain embodiments, the kit further includes a set ofinstructions for practicing methods in accordance with the presentinvention, where the instructions may be associated with a packageinsert and/or the packaging of the kit or the components thereof.

The invention is further illustrated by the following non-limitingexamples.

Reagents

Various reagents are identified in the examples below, the formulationsand pH values (where relevant) of these reagents were as follows.

A “Lysis Buffer” contains 15 mM sodium phosphate monobasic monohydrate,15 mM sodium phosphate dibasic anhydrous, 1.0 mM EDTA disodiumdihydrate, 1.0 mM EGTA free acid, and 110 mM lithium lauryl sulfate, pH6.7.

A “Urine Lysis Buffer” contains 150 mM HEPES free acid, 294 mM lithiumlauryl sulfate, 57 mM lithium hydroxide monohydrate, 100 mM ammoniumsulfate, pH 7.5.

A “Target Capture Reagent” contains 250 mM HEPES free acid dihydrate,310 mM lithium hydroxide monohydrate, 1.88 M lithium chloride, 100 mMEDTA free acid, 2 M lithium hydroxide to pH 6.4, and 250 μg/ml 1 micronmagnetic particles Sera-Mag′ MG-CM Carboxylate Modified (Seradyn, Inc.;Indianapolis, Ind.; Cat. No. 24152105-050450) having oligo(dT)₁₄covalently bound thereto.

A “Wash Solution” contains 10 mM HEPES free acid, 6.5 mM sodiumhydroxide, 1 mM EDTA free acid, 0.3% (v/v) ethyl alcohol absolute, 0.02%(w/v) methyl paraben, 0.01% (w/v) propyl paraben, 150 mM sodiumchloride, 0.1% (w/v) lauryl sulfate, sodium (SDS), and 4 M sodiumhydroxide to pH 7.5.

An “Amplification Reagent” is a lyophilized form of a 3.6 mL solutioncontaining 26.7 mM rATP, 5.0 mM rCTP, 33.3 mM rGTP and 5.0 mM rUTP, 125mM HEPES free acid, 8% (w/v) trehalose dihydrate, 1.33 mM dATP, 1.33 mMdCTP, 1.33 mM dGTP, 1.33 mM dTTP, and 4 M sodium hydroxide to pH 7.5.The Amplification Reagent is reconstituted in 9.7 mL of “AmplificationReagent Reconstitution Solution” described below.

An “Amplification Reagent Reconstitution Solution” contains 0.4% (v/v)ethyl alcohol absolute, 0.10% (w/v) methyl paraben, 0.02% (w/v) propylparaben, 33 mM KCl, 30.6 mM MgCl₂, 0.003% phenol red.

A “Primer Reagent” contains 1 mM EDTA disodium dihydrate, ACS, 10 mMTrizma7 base, and 6M hydrochloric acid to pH 7.5.

An “Enzyme Reagent” is a lyophilized form of a 1.45 mL solutioncontaining 20 mM HEPES free acid dihydrate, 125 mM N-acetyl-L-cysteine,0.1 mM EDTA disodium dihydrate, 0.2% (v/v) TRITON® X-100 detergent, 0.2M trehalose dihydrate, 0.90 RTU/mL Moloney murine leukemia virus(“MMLV”) reverse transcriptase, 0.20 U/mL T7 RNA polymerase, and 4Msodium hydroxide to pH 7.0. (One “unit” or “RTU” of activity is definedas the synthesis and release of 5.75 fmol cDNA in 15 minutes at 37° C.for MMLV reverse transcriptase, and for T7 RNA polymerase, one “unit” or“U” of activity is defined as the production of 5.0 fmol RNA transcriptin 20 minutes at 37° C.) The Enzyme Reagent is reconstituted in 3.6 mLof “Enzyme Reagent Reconstitution Solution” described below.

An “Enzyme Reagent Reconstitution Solution” contains 50 mM HEPES freeacid, 1 mM EDTA free acid, 10% (v/v) TRITON X-100 detergent, 120 mMpotassium chloride, 20% (v/v) glycerol anhydrous, and 4 M sodiumhydroxide to pH 7.0.

A “Probe Reagent” is a lyophilized form of a 3.6 mL solution containing110 mM lithium lauryl sulfate, 10 mM of mercaptoethane sulfonic acid,100 mM lithium succinate, and 3% PVP. The Probe Reagent is reconstitutedin 36 mL of “Probe Reagent Reconstitution Solution” described below.

A “Probe Reagent Reconstitution Solution” contains 100 mM succinic acid,73 mM lithium lauryl sulfate, 100 mM lithium hydroxide monohydrate, 15mM aldrithiol, 1.2 M lithium chloride, 20 mM EDTA, 3% (v/v) ethylalcohol, and 2M lithium hydroxide to pH 4.7.

A “Selection Reagent” contains 600 mM boric acid, ACS, 182.5 mM sodiumhydroxide, ACS, 1% (v/v) TRITON X-100 detergent, and 4 M sodiumhydroxide to pH 8.5.

A “Detection Reagents” comprises Detect Reagent I, which contains 1 mMnitric acid and 32 mM hydrogen peroxide, 30% (v/v), and Detect ReagentII, which contains 1.5 M sodium hydroxide.

An “Oil Reagent” is a silicone oil.

Example 1: S-Complex Preparation

S-Complexes were made in a reagent mixture made up of Target CaptureReagent (minus the magnetic particles), Lysis Buffer and water in a1:2:2 ratio respectively. T7 oligonucleotide, non-T7 oligonucleotide,and S-Oligo were added to the reagent mixture at 6, 5, and 6 picomoles(pmol) per microliter (ul) respectively. The reagent mixture with theoligonucleotides was incubated at 95° C. for one minute in a hot block,followed by a 4° C. incubation for 5 minutes, followed by a roomtemperature incubation for approximately 20 minutes. The S-Complex (1ul) was added to the Target Capture Reagent.

Example 2: Universal Tagged TMA with and without DisplacerOligonucleotides

In this example, several non-T7 amplification oligonucleotidevariations, with and without displacer oligonucleotides were compared.The non-T7 sequence variations were (5′ to 3′): displacertag:spacer:tag:target specific (SEQ ID NO:6); tag:spacer:tag:targetspecific (SEQ ID NO:7); displacer tag:tag:target specific (SEQ ID NO:8);and tag:target specific (SEQ ID NO:9). A T7 promoter-based amplificationoligomer (SEQ ID NO:11) was used with each of the 4 nonT7 amplificationoligomers. The amplification oligomers (nonT7 and T7 pairs) were joinedusing the s-oligo SEQ ID NO:12 (see, e.g., Brentano et al. WO2008/080029 describing reagents and methods for amplification usingforward and reverse amplification oligomers joined with an s-oligo,incorporated herein by reference). The amplification oligonucleotidecombinations were analyzed using target capture to extract targetnucleic acid from a sample; single primer transcription mediatedamplification (spTMA) to amplify the target nucleic acid; and moleculartorches to detect the amplification product (i.e. amplicons) in“real-time” (i.e. continuous monitoring of fluorescent levels overtime). Target Capture is described in Weisburg et al, U.S. Pat. No.6,110,678 (the contents of which are incorporated by reference), spTMAis described in Becker et al., U.S. Pat. No. 7,374,885 and U.S. App. No.20060046265A1 (the contents of which are incorporated by reference), andmolecular torches are described in Becker et al, U.S. Pat. Nos.6,849,412, 6,835,542, 6,534,274, and 6,361,945, US App. No.20060068417A1; and Arnold et al. US App. No. US20060194240A1 (thecontents of which are incorporated by reference). The protocols for eachmethod are briefly described below.

The prostate specific antigen (PSA) gene was cloned and transected intocompetent cells. In vitro transcript (IVT) from the cloned PSA was usedas the target nucleic acid. PSA IVT was spiked into a 1:1 mixture ofLysis Buffer and water at 0, 10.sup.2, and 10.sup.4 copies per 400microliters (ul) and 400 ul of the spiked, diluted Lysis Buffer wastransferred to a 96 deep well plate. Target Capture Reagent (100 ul)containing 5 pmol of SEQ ID NO:1, 1 ul of the S-Complex preparedaccording to Example 1 and using the combinations of nonT7, T7 ands-oligomer described directly above (see Table 1 for S-Complex primercombinations), and 2 pmol of SEQ ID NO:3 blocker was added to each well.The 96 well plate was sealed, a 90° C. heat block was placed on top ofthe plate, and the plate was vortexed on a low setting for 10 seconds.The 96 well plate was incubated at 60° C. for 25 and then cooled to roomtemperature for 25 minutes. The magnetic beads were pelleted using aKingFisher® instrument (Thermo Scientific) and the supernatant wasremoved. The magnetic beads were resuspended in 400 ul of Wash Solution,repelletted, and the wash solution was removed. Following capture andwash, the remaining complex was—a magnetic beadimmobilized probe:captureprobe:target nucleic acid:s-oligo complex according to each of thecombinations in Table 1. The magnetic beads were then resuspended in 60ul of Amplification Reagent containing 10 pmol of SEQ ID NO:4, 15 pmolof each of SEQ ID NO:10, 10 pmol of SEQ ID NO:2 (labeled with ROX andFAM), and for half of the samples, 15 pmol of SEQ ID NO:5. The 96 wellplate was placed in a Chromo4® instrument (Bio-Rad Laboratories, Inc.,Hercules, Calif.) pre-warmed to 42° C. for 5 minutes. Enzyme Reagent (20ul) was added to each well, the plate was briefly vortexed for 20seconds, and returned to the Chromo4 instrument to start thefluorescence monitoring.

Four replicates were run for each assay condition. The results weremeasured by the amount of fluorescence over time. The reported Ct is thecycle time when the fluorescence level becomes higher than thebackground level. The results are summarized in Table 2, below andindicate that the use of tag-mediated displacer technology reduced theaverage Ct for each condition. That is, a fluorescent signal wasdetected earlier when using displacers than when not indicating that theamount of amplification product generated in a condition is greater whenusing the tag-mediated displacer than when not.

TABLE 1 S-Oligomer Complexes T7 SEQ ID NO: Non-T7 SEQ ID NO: S-oligo SEQID NO: 11 6 12 11 7 12 11 8 12 11 9 12

TABLE 2 Amt. of PSA IVT S-Complex Displacer Ave. Ct (minutes) 0 11, 6,12 No No signal 100 11, 6, 12 No 50 10,000 11, 6, 12 No   49.5 0 11, 6,12 Yes No signal 100 11, 6, 12 Yes   41.5 10,000 11, 6, 12 Yes 39 0 11,7, 12 No No signal 100 11, 7, 12 No 45 10,000 11, 7, 12 No 43 0 11, 7,12 Yes No signal 100 11, 7, 12 Yes   42.5 10,000 11, 7, 12 Yes 41 0 11,8, 12 No No signal 100 11, 8, 12 No 47 10,000 11, 8, 12 No 44 0 11, 8,12 Yes No signal 100 11, 8, 12 Yes 45 10,000 11, 8, 12 Yes 41 0 11, 9,12 No No signal 100 11, 9, 12 No   46.5 10,000 11, 9, 12 No 46

Example 3: Sensitivity of Universal Tagged TMA with and withoutDisplacer Oligonucleotides

In this example, the sensitivity of a non-T7 amplificationoligonucleotide (SEQ ID NO:6, displacer:spacer:tag:target specific) withand without displacer oligonucleotides was evaluated. The procedures andoligonucleotide concentrations were the same as those described inExample 2 with the following changes. The PSA IVT was tested at 0, 100,1000, 10000, 100000, and 1000000 copies per reaction. Four replicateswere run for each assay condition. The results are summarized in Table3, below and indicate that the use of displacers reduced the average Ctfor a condition. Tag-mediated displacer technology increases the numberof amplification products generated in a reaction to provide an earlieremergence of fluorescent signal.

TABLE 3 Amt. of PSA IVT Displacer Ct (minutes) 0 No No signal 100 No 601000 No 56 10000 No 50 100000 No 46 1000000 No 39 0 Yes No signal 100Yes 52 1000 Yes 50 10000 Yes 43 100000 Yes 40 1000000 Yes 34

These examples show that tag-mediated displacement increased overalloutput and increased assay kinetics and sensitivity.

TABLE 4 SEQ ID Preferred NO Sequence 5′ → 3′ Function 1CGAACUUGCGCACACACGUCAUUGGAtttaaa Target aaaaaaaaaaaaaaaaaaaaaaaaaaaCapture 2 UGUGUCUUCAGGAUGAAACACACA Torch 3 GAUGCAGUGGGCAGCUGUGAGGABlocker 4 aatttaatacgactcactatagggaga CCACA T7 ACGGTTT 5GAGGTCGTGGCTGGAGTCAT Displacer 6 GAGGTCGTGGCTGGAGTCATatgtcaacgtGT Non-T7CATATGCGACGATCTCAG GCTGTGGCTGACCT GAAATACC 7GTCATATGCGACGATCTCAGatgtcaacgtGT Non-T7 CATATGCGACGATCTCAGGCTGTGGCTGACCT GAAATACC 8 GAGGTCGTGGCTGGAGTCAT GTCATATGCGAC Non-T7GATCTCAG GCTGTGGCTGACCTGAAATACC 9 GTCATATGCGACGATCTCAG GCTGTGGCTGACNon-T7 CTGAAATACC 10 GTCATATGCGACGATCTCAG Non-T7 11aatttaatacgactcactatagggagaCCACA T7 ACGGTTT ACCCAGCAAGATCACGCTTTTG 12AAACCGTTGTGG TCTCCCTATACTGAGATCGT s-oligo CGCATATGAC

Table 4 illustrates oligonucleotide sequences such as those used in theexamples. The legend for Table 4 is as follows. Bold=target specific;Italics=displacer tag; Underline=tag non-T7; Underline italics=tag T7;lowercase=promoter or capture tail.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims. All publications, patents, andpatent applications cited herein are hereby incorporated by reference intheir entireties for all purposes.

1-65. (canceled)
 66. A kit for amplifying a nucleic acid target region,the kit comprising: (1) a first amplification oligomer comprising (a) atarget-binding priming segment (T1) complementary to a 3′-end of thetarget region; and (b) optionally, a heterologous universal tag (U2)located 5′ to T1; whereby the target nucleic acid can serves as atemplate for extension from the first amplification oligomer to producea first amplicon; (2) a second amplification oligomer comprising (a) atarget-binding segment T2 complementary to a region of the firstamplicon that is the complement of a 5′-end of the target region; (b) aheterologous universal tag (U1) located 5′ to T2; and (c) a firstheterologous displacer tag (D1) located 5′ to U1; whereby the firstamplicon can serve as a template for amplification from the secondamplification oligomer to produce a second amplicon comprising U1 andD1; and whereby the second amplicon can serve as a template forextension from the first amplification oligomer to produce a thirdamplicon comprising segments cU1 and cD1, complementary to U1 and D1,respectively; (3) a third amplification oligomer comprising a universalpriming segment U1_(p) having a nucleotide sequence complementary to U1;(4) a fourth amplification oligomer comprising a displacer primingsegment D1_(p) having a nucleotide sequence complementary to D1; and (5)if the first amplification oligomer comprises U2, a fifth amplificationoligomer comprising a second universal priming segment U2_(p) having anucleotide sequence complementary to U2; whereby the third amplicon canserve as a template for extension from both the third and fourthamplification oligomers, wherein extension of U1_(p) from a U1_(p):cU1hybrid produces a fourth amplicon, and wherein extension of D1_(p) froma D1_(p):cD1 hybrid produces a fifth amplicon while displacing thefourth amplicon.
 67. The kit of claim 66, wherein the secondamplification oligomer further comprises an intervening spacer segment(S1) between U1 and D1.
 68. The kit of claim 66, wherein the secondamplification oligomer further comprises a second heterologous displacertag (D2) located 5′ to D1, whereby the second amplicon further comprisesD2 and the third amplicon further comprises segment cD2, complementaryto D2; and wherein the kit further comprises (6) a sixth amplificationoligomer comprising a second displacer priming segment D2_(p) having anucleotide sequence complementary to D2; whereby (i) the third ampliconcan serve as a template for extension from the sixth amplificationoligomer, wherein extension of D2_(p) from a D2_(p):cD2 hybrid producesa sixth amplicon comprising U1, D1, and D2_(p); and (ii) the sixthamplicon can serve as a template for extension from the first or fifthamplification oligomer to produce a seventh amplicon comprising cU1,cD1, and cD2_(p).
 69. The kit of claim 68, wherein the secondamplification oligomer further comprises a second intervening spacersegment (S2) between D1 and D2.
 70. The kit of claim 66, furthercomprising (6) a sixth amplification oligomer comprising (a) primingsegment D1_(p) and (b) displacer tag D2 located 5′ to D1_(p); and (7) aseventh amplification oligomer comprising a second displacer primingsegment D2_(p) having a nucleotide sequence complementary to D2; whereby(i) the fifth amplicon can serve as a template for extension from thefirst or fifth amplification oligomer to produce a sixth ampliconcomprising segments cU2 and cD1_(p); (ii) at least one of the third andsixth amplicons can serve as a template for extension from the sixthamplification oligomer, wherein extension of D1_(p) from a D1_(p):cD1 orD1_(p):cD1_(p) hybrid produces a seventh amplicon comprising U2, D1_(p),and D2; (iii) the seventh amplicon can serve as a template for extensionfrom the first amplification oligomer to produce an eighth ampliconcomprising cU2, cD1_(p), and cD2; (iv) the eighth amplicon can serve asa template for extension from the seventh amplification oligomer,wherein extension of D2_(p) from a D2_(p):cD2 hybrid produces a ninthamplicon comprising U2, D1, and D2_(p); and (v) the ninth amplicon canserve as a template for extension from the first or fifth amplificationoligomer to produce a tenth amplicon comprising cU2, cD1, and cD2_(p).71. The kit of claim 68, further comprising (7) a seventh amplificationoligomer comprising (a) priming segment D1_(p) and (b) displacer tag D2located 5′ to D1_(p); whereby (i) the fifth amplicon can serve as atemplate for extension from the first or fifth amplification oligomer toproduce an eighth amplicon comprising segments cU1 and cD1_(p); (ii) atleast one of the third, seventh, and eighth amplicons can serve as atemplate for extension from the seventh amplification oligomer, whereinextension of D1_(p) from a D1_(p):cD1 or D1_(p):cD1_(p) hybrid producesa ninth amplicon comprising U1, D1_(p), and D2; and (iii) the ninthamplicon can serve as a template for extension from the first or fifthamplification oligomer to produce a tenth amplicon comprising cU1,cD1_(p), and cD2.
 72. The kit of claim 71, wherein the secondamplification oligomer further comprises a third heterologous displacertag (D3) located 5′ to D2, whereby the second amplicon further comprisesD3 and the third amplicon further comprises a segment cD3, complementaryto D3; and the kit further comprises (8) an eighth amplificationoligomer comprising a third displacer priming segment D3_(p) having anucleotide sequence complementary to D3; whereby (i) the third ampliconcan serve as a template for extension from the eighth amplificationoligomer, wherein extension of D3_(p) from a D3_(p):cD3 hybrid producesan eleventh amplicon comprising U1, D1, D2, and D3_(p); and (ii) theeleventh amplicon can serve as a template for extension from the firstor fifth amplification oligomer to produce an twelfth ampliconcomprising cU1, cD1, cD2, and cD3_(p).
 73. The kit of claim 72, whereinthe first amplification oligomer further comprises a third interveningspacer segment (S3) between D2 and D3.
 74. The kit of claim 70, furthercomprising (8) an eighth amplification oligomer comprising (a) primingsegment D2_(p) and (b) displacer tag D3 located 5′ to D2_(p); and (9) aninth amplification oligomer comprising a third displacer primingsegment D3_(p) having a nucleotide sequence complementary to D3; whereby(i) at least one of the eighth and tenth amplicons can serve as atemplate for extension from the eighth amplification oligomer, whereinextension of D2_(p) from a D2_(p):cD2 or D2_(p):cD2_(p) hybrid producesan eleventh amplicon comprising U2, D1/D1_(p), D2_(p), and D3; (ii) theeleventh amplicon can serve as a template for extension from the firstor fifth amplification oligomer to produce a twelfth amplicon comprisingcU2, cD1/cD1_(p), cD2_(p), and cD3; (iii) the twelfth amplicon can serveas a template for extension from the ninth amplification oligomer,wherein extension of D3_(p) from a D3_(p):cD3 hybrid produces anthirteenth amplicon comprising U2, D1, D2, and D3_(p); and (iv) thethirteenth amplicon can serve as a template for extension from the firstor fifth amplification oligomer to produce a fourteenth ampliconcomprising cU2, cD1, cD2, and cD3_(p).
 75. The kit of claim 73, furthercomprising (9) a ninth amplification oligomer comprising (a) primingsegment D2_(p) and (b) displacer tag D3 located 5′ to D2_(p); (i) atleast one of the third, seventh, tenth, and twelfth amplicons can serveas a template for extension from the eighth amplification oligomer,wherein extension of D2_(p) from a D2_(p):cD2 or D2_(p):cD2_(p) hybridproduces a thirteenth amplicon comprising U1, D1/D1_(p), D2_(p), and D3;and (ii) the thirteenth amplicon can serve as a template for extensionfrom the first or fifth amplification oligomer to produce a fourteenthamplicon comprising cU1, cD1/cD1_(p), cD2_(p), and cD3.
 76. The kit ofclaim 75, wherein the first amplification oligomer further comprises afourth heterologous displacer tag (D4) located 5′ to T1, whereby thefirst amplicon comprises T1 and D4; and whereby the second ampliconcomprises segments cT1 and cD4, complementary to T1 and D4,respectively; wherein the kit further comprises (10) a tenthamplification oligomer comprising a priming segment T1_(p) having anucleotide sequence complementary to T1, or complementary to thecomplement of a second amplicon target sequence cT1′ near or overlappingwith cT1 and situated 5′ to cD4; and (11) an eleventh amplificationoligomer comprising a fourth displacer priming segment D4_(p) having anucleotide sequence complementary to D4; and whereby the second ampliconcan serve as a template for extension from both the tenth and eleventhamplification oligomers, wherein extension of T1_(p) from aT1_(p):cT1/cT1′ hybrid produces a fifteenth amplicon, and whereinextension of D4_(p) from a D4_(p):cD4 hybrid produces a sixteenthamplicon while displacing the fifteenth amplicon.
 77. The kit of claim76, wherein the first amplification oligomer further comprises a fourthintervening spacer segment (S4) between T1 and D4.
 78. The kit of claim66, wherein the first amplification oligomer comprises U2 and furthercomprises a fourth heterologous displacer tag (D4) located 5′ to U2,whereby the first amplicon comprises U2 and D4; and whereby the secondamplicon comprises segments cU2 and cD4, complementary to U2 and D4,respectively; wherein the kit further comprises (10) an eleventhamplification oligomer comprising a fourth displacer priming segmentD4_(p) having a nucleotide sequence complementary to D4; and whereby thesecond amplicon can serve as a template for extension from both thefifth and tenth amplification oligomers, wherein extension of U2_(p)from a U2_(p):cU2 hybrid produces a fifteenth amplicon, and whereinextension of D4_(p) from a D4_(p):cD4 hybrid produces a sixteenthamplicon while displacing the fifteenth amplicon.
 79. The kit of claim78, wherein the first amplification oligomer further comprises a fourthintervening spacer segment (S4) between U2 and D4.
 80. The kit of claim66, wherein the affinity of D1_(p) for its complement is lower than thatof U1.
 81. The kit of claim 68, wherein at least one of the followingconditions is present: (a) the affinity of D1_(p) for its complement islower than that of U1; (b) the affinity of D2_(p) for its complement islower than that of D1_(p).
 82. The kit of claim 72, wherein at least oneof the following conditions is present: (a) the affinity of D1_(p) forits complement is lower than that of U1; (b) the affinity of D2_(p) forits complement is lower than that of D1_(p); (c) the affinity of D3_(p)for its complement is lower than that of D2_(p).
 83. (canceled)
 84. Thekit of claim 83, further comprises a reverse transcriptase (RT).
 85. Thekit of claim 84, wherein the first amplification oligomer furthercomprises an RNA polymerase promoter sequence (P) located 5′ to T1,whereby the second amplicon comprises a segment cP, complementary to thepromoter sequence; and whereby an RNA polymerase can initiatestranscription upon recognizing a double-stranded promoter sequence(P:cP) formed by extension of the second amplification oligomer on thefirst amplicon, thereby producing an RNA amplicon. 86-138. (canceled)